Permeation & Gating of Ion Channels

Permeation & Gating of Ion Channels International Workshop, June 20-22, 2014 Strobl am Wolfgangsee, Austria

Abstract Booklet

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

WELCOME

The German Biophysical Society (Deutsche Gesellschaft für Biophysik, DGfB) and the Institute of Biophysics at Johannes Kepler University are very proud and delighted to welcome you to this international workshop on the Permeation and Gating of Ion Channels. This is the third time that the DGfB subgroup “Medical Biophysics” convenes in St. Wolfgang. We thank the German Biophysical Society (DGfB) and the Johannes Kepler University Linz for their financial support. We are also most appreciative for the important contributions from our business sponsors: Nikon, HEKA, Seqlab, Zeiss, Rapp OptoElectronic, Olympus, acal/bfi, Sarstedt, npi, AHF Analysentechnik, Photometrics, Bio Trend and Axon Labortechnik. This meeting was made possible by the dedication of our staff from the Biophysics Institute. A few key players include Anja Engleder who took care of the hotel organization, Quentina Beatty the designer of the conference home page, Lukas Winter the editor of the abstract book, and Andreas Horner, the hike organizer. Finally, we extend a big thank you to all of those who participate in this event. Altogether more than 50 scientists have registered. We hope that you enjoy the presentations and discussions in a serene atmosphere at the foot of the Alps on the beautiful southern shore of the Wolfgangsee. We wish you an enjoyable stay with us and we do hope that it will be a mutually stimulating and successful time together.

Warmest greetings and best wishes,

Johannes Oberwinkler and Peter Pohl

St. Wolfgang, 20 June 2014

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TABLE OF CONTENTS Page Area Map ........................................................................................ 3 Program Overview .......................................................................... 4 Program .......................................................................................... 5 Abstracts....................................................................................... 11 Poster Abstracts ........................................................................... 38 Participants ................................................................................... 56

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AREA MAP

The workshop is located at the bifeb (red).

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PROGRAM OVERVIEW Friday, June 20th 2014

Registration Afternoon Sessions:

TRP CHANNEL PHARMACOLOGY PERMEATION AT THE SELECTIVITY FILTER

Plenary Evening Talk:

BK CHANNEL GATING (RAMON LATORRE)

Saturday, June 21st 2014

Morning Session:

GATING OF TRP CHANNELS INTERACTION OF CHANNELS

Hiking Trip Plenary Evening Talk:

VOLTAGE SENSOR CONFORMATIONAL CHANGES (FRANCISCO BEZANILLA)

Sunday, June 22nd 2014

Morning Session:

MODELLING AND THEORY OF PERMEATION THERMO- AND NOCICEPTIVE TRP CHANNELS

Closing

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PROGRAM Friday, June 20th, 2014 Registration / Rooms 12.00 – 13 .00

Lunch

TRP CHANNEL PHARMACOLOGY 13.00 – 13.40 13.40 – 14.20 14.20 – 14.50

Michael Schaefer (Leipzig) Targeting TRP channels with small molecules

Thomas Gudermann (München) TRP channels and magnesium homeostasis

Michael G. Leitner (Marburg) TRPM4 channel activation by covalent modification

PERMEATION AT THE SELECTIVITY FILTER Arin Marchesi (Trieste) 14.50 – 15.20

Mechanism of ionic permeation in the mimics of CNG channels: A structural, functional and computational analysis

15.20 – 15.50

Coffee Break

15.00 – 15.40

Crina Nimigean (New York)

15.40 – 16.20

Molecular locations of gates in MthK potassium channels

György Panyi (Debrecen) Slow inactivation of Shaker K+ channels: gates, states and transitions

16.20 – 16.40

Peter Pohl (Linz) Water transport through potassium channels

18.00 – 19.00

Dinner

PLENARY EVENING TALK Ramon Latorre (Valparaíso) 19.00 – 20.00

Gating and Architecture of the BK Channel in the Presence of Auxilliary β Subunits

20.00 – open end

POSTERS Beer and Wine 5

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Saturday, June 21st, 2014 08.00 – 09.00

Breakfast

GATING OF TRP CHANNELS 08.30 – 09.10

Johannes Oberwinkler (Marburg) Regulation of TRPM3 activity

Klaus Groscher (Graz) 09.10 – 09.40 09.40 – 10.20 10.20 – 10.50

Novel insight into the lipid-gating machinery of TRPC3 - a structure guided mutagenesis approach

Roger Hardie (Cambridge) TRP channels and phototransduction in Drosophila

Coffee Break

INTERACTION OF CHANNELS 10.50 – 11.20

Simon Scheuring (Marseille) High-speed AFM monitors membrane protein dynamics

Max Ulbrich (Freiburg) 11.20 – 11.50

Dissecting subunit assembly by single molecule imaging of membrane protein complexes

Rainer Schindl (Linz) 11.50 – 12.20

Novel trans-membrane mutation switches Orai1 to a constitutively active and Ca2+ selective channel

Rudolf Schubert (Heidelberg) 12.20 – 12.50

Kv channel activity determines the functional availability of smooth muscle BK channels in intact arteries

12.50 – 13.45

Lunch

13.45 – 18.00

Hiking

18.00 – 19.00

Dinner

PLENARY EVENING TALK 19.00 – 20.00 20.00 – open end

Francisco Bezanilla (Chicago) Voltage sensor conformational changes

POSTERS Beer and Wine 6

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Sunday, June 22nd, 2014 07.30 – 08.30

Breakfast

MODELLING AND THEORY OF PERMEATION 08.30 – 09.10 09.10 – 09.30

Jochen Hub (Göttingen) Energetics of Solutes at Membranes and Interfaces

Nicolas Wieder (Heidelberg) Fully Stochastic Modelling of the IP3 Receptor Channel

Stefan Howorka (London) 09.30 – 10.00

Membrane-Spanning DNA Nanopores: Biomimetic Chemical Structures for Single-Molecule Research and Nanotechnology

Petronel Tuluc (Innsbruck) 10.00 – 10.20

Residues critical for voltage-sensor transitions determining gating properties of CaV1.1

10.20 – 10.50

Coffee Break

THERMO- AND NOCICEPTIVE TRP CHANNELS Peter McNaughton (London) 10.50 – 11.30 11.30 – 12.10

Modulation of TRP channels - fundamental mechanisms and in vivo significance

Thomas Voets (Leuven) Dissecting and disturbing thermosensitive TRP channels

Katharina Held (Leuven) 12.10 – 12.30 12.30 – 12.50

A single agonist competent to open multiple ion permeation pathways in the nociceptor TRPM3

Marc Behrendt (Marburg) Differential regulation of TRPM3 splice variants

12.50 – 13.00

Closing

13.00 – 14.00

Lunch End of Meeting and Departure

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Posters Lucía Alonso-Carbajo (Leuven) 1

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells

Sandeep Dembla (Marburg) 2

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Role of transient receptor potential melastatin 3 channels in the peripheral nervous system

Liudmila Erokhova (Linz) Water transport through the sodium-glucose cotransporter SGLT1

Christian Goecke (Marburg) 4

Structural requirements for agonism at TRPM3 cation channels

Christian Halaszovich (Marburg) 5

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Engineered Voltage Sensing Phosphatases: What do they tell us about the gating mechanism?

Andreas Horner (Linz) Mobility of water in a confined proteinaceous environment

Denis Knyazev (Linz) 7

Voltage mediated gating of the bacterial protein translocation channel SecYEG

Lubica Lacinova (Bratislava) 8

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Gating of the CaV3.3 channel differs from gating of CaV3.1 and CaV3.3 channels

Andreas Lieb (Innsbruck) Molecular gating mechanism of CaV1.3 voltage gated calcium channels

Nadine J. Ortner (Innsbruck) 10

Pyrimidine 2, 4, 6-triones are a new class of voltage-gated L-type Ca2+ channel activators

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Elena Pohl (Wien) 11

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Impact of different aldehydes on the biophysical parameters of lipid bilayers reconstituted with UCP1

Alicia Sánchez Linde & Brett Boonen (Leuven) Cinnamaldehyde modulates capsaicin-evoked TRPV1 activation

Franziska Schneider (Marburg) 13

TRPM1 channel activity is affected by mutations and G-protein coupled receptors

Azmat Sohail (Wien) 14

Decrypting the Structure of LeuTAa Employing Luminescence Resonance Energy Transfer (LRET)

Balász I. Tóth (Leuven) 15

Cellular regulation of transient receptor potential melastatin 3 (TRPM3) channel activity

Bettina Wilke (Marburg) 16

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A six Amino Acid Motif in the Proximal C-Terminus of TASK-3 Channels Conveys Diacylglycerol-mediated Inhibition

Lukas Winter (Linz) YidC forms a protein translocating channel

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We gratefully acknowledge the generous support from our sponsors:

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ABSTRACTS Friday, June 20th, 2014 Registration / Rooms 12.00 – 13 .00

Lunch

TRP CHANNEL PHARMACOLOGY 13.00 – 13.40 13.40 – 14.20 14.20 – 14.50

Michael Schaefer (Leipzig) Targeting TRP channels with small molecules

Thomas Gudermann (München) TRP channels and magnesium homeostasis

Michael G. Leitner (Marburg) TRPM4 channel activation by covalent modification

PERMEATION AT THE SELECTIVITY FILTER Arin Marchesi (Trieste) 14.50 – 15.20

Mechanism of ionic permeation in the mimics of CNG channels: A structural, functional and computational analysis

15.20 – 15.50

Coffee Break

15.00 – 15.40

Crina Nimigean (New York)

15.40 – 16.20

Molecular locations of gates in MthK potassium channels

György Panyi (Debrecen) Slow inactivation of Shaker K+ channels: gates, states and transitions

16.20 – 16.40

Peter Pohl (Linz)

18.00 – 19.00

Dinner

PLENARY EVENING TALK Ramon Latorre (Valparaíso) 19.00 – 20.00

Gating and Architecture of the BK Channel in the Presence of Auxilliary β Subunits

20.00 – open end

POSTERS Beer and Wine 11

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Targeting TRP channels with small molecules N. Urban, I. Straub, L. Wang, K. Hill, S.E. Philipp, U. Krügel, S. Neuser, W.M. Kübler, J. Teichert, F. Mohr, M. Konrad, O. Rizun, J. Oberwinkler, M. Schaefer Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Härtelstr. 16-18, 04107 Leipzig

Pharmacological modulation of TRP channels is a still underdeveloped field. Poorly specific historic TRP channel modulators, such as fenamates, SK&F96365, 2-APB or lanthanides have caused a significant confusion in the field. Selectively acting tool compounds are urgently needed to acutely modulate TRP channel functions and to explore their therapeutic potential in diseased states. Despite increasing evidence for the involvement of TRP channels in disease-related homeostatic functions, activities in the private sector have initially been capitalized on the targeting of nociceptive sensory TRP channels. To identify, validate and further optimize drug-like compounds that target other TRP channel isotypes, we have set up an academic screening environment and performed medium-throughput compound screens for several TRPC and TRPM channels. The most promising hits were achieved with TRPC6 and TRPM3. TRPM3, recently found to be a heat-activated channel expressed in nociceptive neurons, is potently inhibited by flavanones. With an IC50 of 80 nM, isosakuranetin potently and efficiently suppresses TRPM3-triggered Ca2+ entry and ionic currents in vitro, and TRPM3-related pain responses in vivo. Of note, isosakuranetin neither affected other sensory TRP channels that are expressed in dorsal root ganglion neurons, nor led to increases in the body temperature – a major drawback that is associated with inhibition of TRPV1. Targeting of TRPC6 was initially achieved with a modestly potent and TRPC6-prevalent aryl-b-carboline compound. Inhibition of hypoxia-induced pulmonary vasoconstriction (HPV) in an isolated perfused mouse lung model by the compound mimicked the phenotype of TRPC6-deficient mice. In another approach, we identified a conifer turpentine ingredient, which more potently and selectively suppresses TRPC6, compared to its closest relative TRPC3. This compound was found to be enriched in larch resin, and was identified as larixyl acetate. Larixyl acetate exhibits an IC50 of 100-500 nM, a more than 10-fold selectivity for TRPC6 compared to TRPC3, reversibly inhibits TRPC6 currents, and is biologically active with regard to TRPC6-related Ca2+ responses in pulmonary artery smooth muscle cells, as well as in the mouse HPV model. Validated and acutely acting TRP channel modulators are expected to become valuable tools to establish experimental therapies and thereby to clarify the importance of TRP channels in diseased states. Eventually, these biologically active molecules may be further developed into lead compounds for the treatment of human TRP-channel-related diseases.

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TRP channels and magnesium homeostasis Vladimir Chubanov1, Attila Braun2,3, Bernhard Nieswandt2,3 and Thomas Gudermann1 1

Walther-Straub-Institut of Pharmacology and Toxicology, University of Munich, Germany 3 Department of Vascular Medicine, University Hospital, Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Germany 2

Magnesium is the most abundant divalent cation in living cells and virtually every biological process, e.g. enzyme and membrane function, maintenance of protein structure, ion channel regulation, requires this cation. Magnesium deficiency is associated with human diseases such as eclampsia, metabolic syndrome, type 2 diabetes and cardiovascular disease (hypertension, arrhythmia, atherosclerosis, myocardial infaction). However, the molecular mechanisms underlying organismal magensium homeostasis are only poorly understood. More than 15 magnesium permeable ion channels and transporters have been described whose biological significance, however, still remains largely obscure. Taking advantage of naturally occurring mutations in humans and several genetically engineered mouse models, we identify the Transient Receptor Potential (TRP) ion channel TRPM6 as a master regulator of organismal magnesium homeostasis essential for embryonic development, postnatal survival, aging and longevity. TRPM6 and its close relative TRPM7 are bifunctional proteins comprising a TRP channel segment linked to an α-type protein kinase. At present, the biological significance of such a combination of channel and kinase moieties in one protein is not understood and distinct physiological roles that can be ascribed either to channel or to kinase function have not been delineated. A mutant mouse strain expressing a kinase-dead TRPM7 protein allows a first glance at defined cellular functions of channel-linked -kinases.

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TRPM4 Channel Activation by Covalent Modification Michael G. Leitner*, Niklas Michel, Moritz Lindner, Marc Behrendt, Johannes Oberwinkler and Dominik Oliver Institute of Physiology, Department for Neurophysiology, Philipps-University Marburg, Germany *Correspondence to [email protected]

TRPM4 (transient receptor potential melastatin 4) channels constitute Ca2+-activated nonselective cationic currents in a variety of tissues including heart and brain, and in cells of the immune system (1-3). The channels are gated by elevated cytosolic Ca2+, and activity is regulated by signalling molecules such as ATP and phospholipids (e.g. PI(4,5)P2) (1, 5, 6). Activation of TRPM4 depolarises the membrane potential and modulates the driving force for ions and the activity ion channels (4). TRPM4 agonists and Ca2+-independent gating have not been demonstrated yet. In patch clamp experiments, we found that phospholipase C (PLC) inhibitor U73122 activated recombinant human TRPM4 and endogenous TRPM4 channels in cell lines (6). TRPM4 current amplitudes were independent on PI(4,5)P2 levels arguing against an involvement of PLC inhibition (6). Strikingly, U73122 activated TRPM4 even in the absence of intracellular Ca2+. As the structural analogue U73343 that lacks the active maleimide group was ineffective, these findings suggested covalent modification of TRPM4. In fact, pre-application of N-ethylmaleimide (NEM) preventing further covalent modification abolished the activation of TRPM4 by U73122. Since the small molecule NEM did not activate TRPM4, conjugation of bulkier substances might be required for augmentation of TRPM4 activity. Indeed, TRPM4-mediated currents were potentiated by an inflammatory prostaglandin (15d-PGJ2) that has been shown to activate TRPA1 analogously (7). Taken together, we demonstrated activation of otherwise Ca2+-dependent TRPM4 channels through covalent modification. These findings propose endogenous agonists as physiologically relevant TRPM4 activators. This work was supported by Deutsche Forschungsgemeinschaft through SFB 593 (TP A12) to D.O. and by a Research Grant of the UKGM to M.G.L. References 1. Launay, P., A. Fleig, A. L. Perraud, A. M. Scharenberg, R. Penner, and J. P. Kinet. 2002. TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 109:397-407. 2. Guinamard, R., M. Demion, and P. Launay. 2010. Physiological roles of the TRPM4 channel extracted from background currents. Physiology (Bethesda) 25:155-164. 3. Vennekens, R., and B. Nilius. 2007. Insights into TRPM4 function, regulation and physiological role. Handbook of experimental pharmacology:269-285. 4. Launay, P., H. Cheng, S. Srivatsan, R. Penner, A. Fleig, and J. P. Kinet. 2004. TRPM4 regulates calcium oscillations after T cell activation. Science 306:1374-1377. 5. Nilius, B., J. Prenen, J. Tang, C. Wang, G. Owsianik, A. Janssens, T. Voets, and M. X. Zhu. 2005. Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4. The Journal of biological chemistry 280:6423-6433. 6. Nilius, B., F. Mahieu, J. Prenen, A. Janssens, G. Owsianik, R. Vennekens, and T. Voets. 2006. The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate. The EMBO journal 25:467-478. 7. Takahashi, N., Y. Mizuno, D. Kozai, S. Yamamoto, S. Kiyonaka, T. Shibata, K. Uchida, and Y. Mori. 2008. Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators. Channels (Austin) 2:287-298.

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Mechanism of ionic permeation in the mimics of CNG channels: A structural, functional and computational analysis Luisa M. R. Napolitano1,2, Manuel Arcangeletti1, Ina Bisha1, Matteo De March2, Arin Marchesi1, Silvia Onesti2, Alessandro Laio1, and Vincent Torre1,* 1

Neurobiology Sector, International School for Advanced Studies (SISSA), Trieste 34136, Italy. Structural Biology Laboratory, Elettra-Sincrotrone Trieste, Area Science Park, Basovizza, Trieste 34149, Italy.*Correspondence to [email protected] 2

Using an engineered cyclic nucleotide-gated (CNG) channel mimics based on the bacterial nonselective NaK channel [1], we examined how different ionic species interact with the pore using electrophysiology, all-atoms molecular dynamics simulations and X-ray crystallography. When the mimics is crystallized in the presence of Li+, Na+, K+, Rb+, Cs+ and methylamonium (MA+) the backbone of the pore region and the binding sites show a high degree of flexibility. The side chains of Glu66 and Thr67, located at the extracellular entrance of the selectivity filter show multiple conformations. In the presence of MA+, Glu66 points towards Tyr55 of the same subunit, while in the presence of K+, Glu66 forms H-bonds with Thr60 of a neighboring subunit [1]. Large-scale molecular dynamics simulations in a hydrated lipid bilayer at 0 mV indicate that Glu66 side chains are capable of large structural fluctuations which are modulated by the nature of the ion residing in the pore. In particular, in presence of Cs+ Glu66 explores at least three different conformations, characterized by a significantly different distribution of the local charge. Single-channel recordings from this chimeric ion channel in symmetrical Cs+ conditions show a strong voltage dependency similar to what observed in the native CNGA1 channel [2]. Our results indicate that the selectivity filter of CNG channels has a dynamic structure capable of fine structural rearrangements. Membrane voltage catalyzes conformational changes in the pore region which depend on the permeating ion. References [1] Derebe, M.G., W. Zeng, Y. Li, A. Alam, and Y. Jiang. 2011. Structural studies of ion permeation and Ca2+ blockage of a bacterial channel mimicking the cyclic nucleotide-gated channel pore. Proc. Natl. Acad. Sci. U. S. A. 108: 592–597. [2] Marchesi, A., M. Mazzolini, and V. Torre. 2012. Gating of cyclic nucleotide-gated channels is voltage dependent. Nat. Commun. 3: 973.

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Molecular locations of gates in MthK potassium channels Crina Nimigean Department of Physiology and Biophysics, Cornell University, New York, USA

Understanding how ion channels open and close their pores is crucial for understanding their physiological roles. We used intracellular quaternary ammonium blockers to locate the voltage and calcium-dependent gates in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology, stopped-flow spectrofluorometry, and X-ray crystallography. Blockers bind in an aqueous cavity between two putative gates, an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location: an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. A kinetic single-channel analysis of tetrabutylammonium block of MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutylantimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggests that MthK gating with voltage also leads to conformational changes within the cavity and intracellular pore entrance. For locating the calcium gate, we employed a Tl+ flux assay using a stopped-flow spectrofluorometer. MthK channel activity was estimated from the rate of a MthK-containing liposome-trapped fluorophore quenching due to Tl+ influx through the channels. The high-affinity blocker tetrapentylammonium, applied prior to channel activation using a sequential mixing protocol, was able to fully block closed channels, indicating that the blocker can reach its binding site in closed channels. Given that the blocker binding site is in the cytoplasmic access below the selectivity filter, these results suggest that there is also a calcium-dependent gate at the selectivity filter in MthK.

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Slow inactivation of Shaker K+ channels: gates, states and transitions Gyorgy Panyi*, Zoltan Varga, Florina Zakany, Tibor G. Szanto Department of Biophysics and Cell Biology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary *Correspondence to [email protected]

In the absence of N-type inactivation Shaker potassium channels display slow (C-type) inactivation. The slow-inactivation gate (I-gate) is in the selectivity filter near the extracellular entrance of the pore whereas the activation gate (A-gate), formed by the bundle crossing of the S6 segments, is near the intracellular entrance of the pore (Fig. 1, left panel). The positions of the two gates define four composite gating states of the channel as depicted in right panel of Fig. 1.

Functional and structural studies show that these two gates are coupled. Transitions between the C and O states have been extensively studied previously, whereas the transitions with closed inactivation cannot be studied using conventional electrophysiological methods as these states are non-conducting ones. We have addressed the transitions in the inactivated state using a state dependent cysteine accessibility strategy were cysteines are engineered into strategic positions in the channel protein and their accessibility is measured by the current loss in the presence of MTS reagents and/or Cd2+ in a state dependent manner. Inside/out patches were pulled from tsA201 cells expressing Shaker fast inactivation removed (IR) channels containing T449A mutation to facilitate slow inactivation along with cysteine substitutions at various positions along S6. Using an ultrafast perfusion system we applied the cysteine reagents selectively to the closed, open or inactivated channels (OI and CI). Using this strategy we have reached the following conclusions: 1, The pattern of the accessibility of the cysteins in S6 (470C-474C) indicates that the S6 helix makes rotational motion around its own axis during the direct O→OI transition ensuring the coupling between the activation and inactivation gates. 2, Cd2+ accessibility of T449A/V474C Shaker-IR channels reported that at fairly negative holding potentials, at which no macroscopic current can be detected, rare channel openings occur yielding access to the channel cavity. These rare openings facilitate inactivation of the channels and may explain the apparent “closed state inactivation” reported in classical electrophysiological studies. Based on Cd 2+ accessibility we suggest that A-gate opening precedes structural changes associated with slow inactivation. 3, It has been shown earlier that Cd2+ traps the V476C Shaker channels in the open state, even at very negative voltages, by forming a metal bridge between a cysteine in one subunit and a native histidine (H486) in a neighboring subunit. We used this construct to determine if locking the activation gate in the open configuration prevents recovery from inactivation. Our results showed the lack of recovery from inactivation of the locked-open channels thereby suggesting that losure of the activation gate is essential for the recovery from slow inactivation. In summary, we have provided experimental proof for the existence of some hypothetical gating transitions in the inactivated state proposed several decades ago and also proposed that S6 may couple the activation and inactivation gates in Shaker K+ channels. Supported by OTKA K75904 and TÁMOP-4.2.2-A-11/1/KONV-2012-0025

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Water Flow through K+ Channels Peter Pohl Institute of Biophysics, [email protected]

Johannes

Kepler

Universität

Linz,

Gruberstr. 40,

4020 Linz,

Austria;

The selectivity filter of K+ channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K+ hydration. Equilibrium measurements suggested that the resulting structure binds K+ with micromolar affinity. In order to explain K+ turnover numbers of ~108 s-1, the equilibrium affinity is purported to diminish by three or four orders of magnitude due to an electrostatic destabilization that originated from a second ion entering the filter. However, this knock-on mechanism is not compatible with observations which established that osmotic water flow knocks all ions out of the channel (Saparov and Pohl, 2004). Here we tested whether both the inactivated and the non-inactivated (collapsed) conformation of the selectivity filter are water permeable. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K+ channel KcsA into lipid vesicles that were either C-type inactivating or non-inactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. With the aid of stopped-flow experiments, quantification of the per-channel increment in proteoliposome water permeability yielded a unitary water permeability pf of (6.9±0.6)x10-13cm3s-1 for both mutants. “Collapse” of the selectivity filter upon K+ removal did not alter pf and was fully reversible, as demonstrated by current measurements through planar bilayers in a K+-containing medium to which K+-free proteoliposomes were fused. Water flow through KcsA is halved by 200mM K+ in the aqueous solution, which, for a singly occupied channel indicates an effective K+ dissociation constant in that range. This casts doubt on the widely accepted hypothesis that multiple K+ ions in the selectivity filter act to mutually destabilize binding (Hoomann et al., 2013). Preliminary experiments suggest that eukaryotic voltage-sensitive channels share this water conducting feature with the prokaryotic KcsA. References: Saparov, S.M., and Pohl, P. (2004). Beyond the diffusion limit: Water flow through the empty bacterial potassium channel. Proc. Natl. Acad. Sci. U. S. A. 101, 4805-4809. Hoomann, T., Jahnke, N., Horner, A., Keller, S., and Pohl, P. (2013). Filter gate closure inhibits ion but not water transport through potassium channels. Proc. Natl. Acad. Sci. U. S. A. 110, 10842-10847.

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Strobl, June 20-22, 2014

Gating and Architecture of the BK Channel in the Presence of Auxilliary β Subunits Ramon Latorre Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile

Calcium- and voltage-activated potassium channels (BK) are regulated by a multiplicity of signals. The prevailing view is that different BK gating mechanisms concur to determine channel opening and that these gating mechanisms are allosterically coupled. In most instances the pore forming α subunit of BK is associated to one of four alternative β subunits that appear to target specific gating mechanisms to regulate the channel activity. In particular, β1 stabilizes the active configuration of the BK voltage sensor, which affects channel Ca2+ sensitivity. To determine the extent to which β subunits regulates the BK voltage sensor, we [1] measured gating currents induced by the pore-forming BK α subunit alone and with the different β subunits expressed in Xenopus oocytes (β1, β2IR, β3b, and β4). We found that β1 and β2 stabilize the BK voltage sensor in the active conformation. However, β3b stabilize the resting configuration of the voltage sensor. In addition, β4 decreases the apparent number of charges per voltage sensor. The decrease in the charge associated with the voltage sensor in α/β4 channels explains most of these channels' biophysical properties. For channels composed of the α-subunit alone, gating charge increases slowly with pulse duration as expected if a significant fraction of charge develops with a time course comparable to that of K+ current activation. Because in the presence of β1, β2 and β4 this slow component develops in advance of and much more rapidly than ion current activation it is possible to appreciate that BK channel opening proceeds in two steps. β subunits are membrane proteins consisting of two putative transmembrane regions connected by a large extracellular loop. In order to reveal the molecular regions in these β subunits that determine their differential functional coupling with the pore-forming α subunit, we made chimeric constructs between β1 and β3 subunits and we measured the gating currents induced by them. Chimeric exchange of the different regions of the β1 and β3 subunits demonstrates that the NH3 terminal is the most relevant region in defining the behavior of either subunit. This strongly suggests that this intracellular domain is crucial for the fine tuning of the effects of these β subunits. In spite of the proven importance of this channel, little is known about its detailed structure. Using lanthanide-based resonance energy transfer (LRET) combined with symmetric nano-positioning system (SNPS) analysis, we have determined the external architectural details of BK channels. We used a genetically encoded lanthanide binding tag (LBT) to bind Tb3+ as LRET donor and a fluorophore-labeled scorpion toxin, iberiotoxin (IbTX), as the LRET acceptor for in vivo measurement of intramolecular distances within the BK channel structure. By introducing LBTs in the extracellular region of the α or β 1 subunit we determined (i) a basic extracellular map of the BK channel, (ii) β 1 subunit-induced rearrangements in α subunits, and (iii) the relative position of the β 1 subunit within the α/β1 complex. Our results provide first glimpses at the BK channel’s external surface structure in its different functional states with and without the β1 subunit, and indicate that β1 most strongly interacts with the α S0 segment. References [1] Contreras, GF, Neely, A., Alvarez, O, Gonzalez, C, and Latorre, R. 2012. Modulation of BK Channel Voltage Gating by Different Auxiliary β Subunits- Proc. Natl. Acad. Sci. (USA). 109:18577-18582.

19

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Saturday, June 21st, 2014 08.00 – 09.00

Breakfast

GATING OF TRP CHANNELS 08.30 – 09.10

Johannes Oberwinkler (Marburg) Regulation of TRPM3 activity

Klaus Groscher (Graz) 09.10 – 09.40 09.40 – 10.20 10.20 – 10.50

Novel insight into the lipid-gating machinery of TRPC3 - a structure guided mutagenesis approach

Roger Hardie (Cambridge) TRP channels and phototransduction in Drosophila

Coffee Break

INTERACTION OF CHANNELS 10.50 – 11.20

Simon Scheuring (Marseille) High-speed AFM monitors membrane protein dynamics

Max Ulbrich (Freiburg) 11.20 – 11.50

Dissecting subunit assembly by single molecule imaging of membrane protein complexes

Rainer Schindl (Linz) 11.50 – 12.20

Novel trans-membrane mutation switches Orai1 to a constitutively active and Ca2+ selective channel

Rudolf Schubert (Heidelberg) 12.20 – 12.50

Kv channel activity determines the functional availability of smooth muscle BK channels in intact arteries

12.50 – 13.45

Lunch

13.45 – 18.00

Hiking

18.00 – 19.00

Dinner

PLENARY EVENING TALK 19.00 – 20.00 20.00 – open end

Francisco Bezanilla (Chicago) Voltage sensor conformational changes

POSTERS Beer and Wine 20

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Regulation of TRPM3 activity Florian Mohr1, Christian Geocke1, Anne Vogel1, Ina Eisenbach1, Sandeep Dembla1, Marc Behrendt1 and Johannes Oberwinkler1* 1

Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Marburg, Germany *Correspondence to [email protected]

The Ca2+-permeable channels formed from TRPM3 proteins can be activated by various chemical substances, including the steroid pregnenolone sulfate and the dihydropyridine nifedipine (1). Studies using structural homologs of these compounds and modified TRPM3 proteins with various point mutations demonstrated that steroids and dihydropyridines do not bind to the same binding site while activating TRPM3 channels. Rather, these substances synergistically activate TRPM3 (2). Recently, also potent inhibitors of TRPM3 activity have been identified (3). Although pregnenolone sulfate is naturally produced in mammals, the high concentrations (micromolar range) typically needed for activating TRPM3 channels raises the question whether TRPM3 activity is regulated by this substance under physiological conditions. Therefore, the physiologically relevant stimuli regulating TRPM3 activity still are not well established. Working with pancreatic β cells (which endogenously express TRPM3 channels), we show that the activation of α2-adrenoreceptors strongly suppresses TRPM3 activity. This effect appears to be due to direct inhibition of TRPM3 channels by G-protein β/γ subunits. We describe the structural requirements necessary for this interaction. The fast and potent nature of TRPM3 inhibition through a well-known signal transduction cascade indicates that this regulation is physiologically important. References

1.

2.

3.

Wagner, T. F., S. Loch, S. Lambert, I. Straub, S. Mannebach, I. Mathar, M. Düfer, A. Lis, V. Flockerzi, S. E. Philipp, and J. Oberwinkler. 2008. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells. Nat Cell Biol 10:1421-1430. Drews, A., F. Mohr, O. Rizun, T. F. Wagner, S. Dembla, S- Rudolph, S. Lambert, M. Konrad, S. E. Philipp, M. Behrendt, S. Marchais-Oberwinkler, D. F. Covey and J. Oberwinkler. 2014. Structural requirements of steroidal agonists of transient receptor potential melastatin 3 (TRPM3) cation channels. Br J Pharmacol 171:1019-1032. Straub, I., U. Krügel, F. Mohr, J. Teichert, O. Rizun, M. Konrad, J. Oberwinkler, M. Schaefer. 2013. Flavanones that selectively inhibit TRPM3 attenuate thermal nociception in vivo. Mol Pharmacol 84:736-750.

21

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Novel insight into the lipid-gating machinery of TRPC3 - a structure guided mutagenesis approach Klaus Groschner and Michaela Lichtenegger Institut für Biophysik, Medizinische Universität Graz, Austria

TRPC3 is a lipid-sensitive member of the family of canonical transient receptor potential channels that is likely involved in maladaptive cardiovascular remodeling and neurodegenerative disorders. Consequently, a better understanding of the channel´s gating and its control by membrane lipids and/or small-molecular modulators is expected to enable development of novel therapeutic strategies. Guided by a homology model of TRPC3 architecture, based on the structural information of its relative TRPV1, mutagenesis experiments were performed to identify molecular elements that determine lipid-sensitivity and gating of TRPC3. Mutations in a hydrophobic cluster at the Cterminal end of transmembrane domain 6, corresponding to the S6 helix bundle-crossing region (BC-gate) in KV channels, affected constitutive activity. Destabilization of this potential BC-gate by replacing a large hydrophobic residue (I667A) was not only associated with profound constitutive activity but also with significant changes in ion selectivity as well as blocker sensitivity, indicating a strict dependency of the selectivity filter (SF) at the pore entrance on gating status. Structure-guided mutagenesis further identified a glycine residue (G652) located between the hypothetical SF and BC-gate structures as a critical determinant of TRPC3 function. Mutations in this residue were found to selectively affect channel activation in response to phospholipase C activity, diacylglycerols and a novel synthetic activator of TRPC3. A structural model of the TRPC3 permeation pathway is presented localizing the channel’s selectivity filter as well as physical gate. We provide evidence for coupling between occluding gate and selectivity filter, and suggest involvement of at least two distinct gating motions in TRPC3 activation.

22

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

TRP channels and phototransduction in Drosophila Roger Hardie Cambridge University Department of Physiology Development and Neuroscience

As in other microvillar photoreceptors, phototransduction in Drosophila is mediated by a Gprotein coupled phospholipase C (PLC) cascade, culminating in activation of “transient receptor potential” (TRP) channels. Incident light is absorbed and transduced in the rhabdomere, a 1-2 μm diameter light-guiding stack of tightly packed microvilli, containing the visual pigment rhodopsin and other major components of the cascade. How PLC leads to activation of TRP channels remains controversial. PLC hydrolyses the membrane phospholipid, PIP2, to generate DAG, InsP3 and a proton. In addition, hydrolysis of PIP2’s bulky inositol headgroup from the inner leaflet of the lipid bilayer should reduce membrane area, thereby increasing membrane tension, leading to contraction of the microvilli. Recent results suggest that light-sensitive TRP channels may be gated by a combinatorial mechanism involving mechanical forces in the microvillar membrane bilayer resulting from PIP2 depletion, acting in concert with the protons released by PLC.

23

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

High-speed atomic force microscopy (HS-AFM) monitors membrane protein dynamics Simon Scheuring1*, Ignacio Casuso1, Adai Colom1, Felix Rico1 1

U1006 INSERM, Université Aix-Marseille, Campus Luminy, 13009 Marseille, France *Correspondence to [email protected]

Membrane-mediated protein-protein and protein-lipid interactions, membrane protein localization, and related dynamics, modulate membrane protein function [1]. So far membrane structure and dynamics could not be studied altogether lacking the technique that analyses unlabelled proteins at submolecular lateral and high temporal resolution. Here we used high-speed atomic force microscopy (HS-AFM, [2]) to characterize the movements and interactions of unlabelled porin OmpF [3] and aquaporins [4] in native membranes. First an introduction to AFM and its use in membrane biology will be given, followed by an introduction to the membrane proteins and the membrane structure as a current challenge in biology. Using HS-AFM, we are able to describe essential novel aspects that govern membrane protein assembly and membrane superstructure. Protein motion scales roughly with membrane crowding. However molecules display individuality of diffusion behaviour ranging from fast moving to immobile molecules trapped by favourable protein-protein associations. We derive the molecular interaction probability landscapes and assembly rationales that we compare with coarse-grained molecular dynamics and Monte Carlo simulations. Most recently, we were able to monitor for the first time individual unlabelled membrane proteins directly on cells [5]. HS-AFM may open a novel research avenue that bridges structure of individual membrane proteins and supramolecular membrane architecture. Finally, high-speed force spectroscopy (HS-FS) allows the characterization of interactions within and between molecules over a wide dynamic range [6]. References [1] D.M. Engelman, Nature 438, 578 (2005) [2] T. Ando, et al., Proceedings of the National Academy of Sciences 98, 12468 (2001) [3] I. Casuso, et al., Nature Nanotechnology, 7, 525 (2012) [4] A. Colom, et al., Journal of Molecular Biology, 423, 249 (2012) [5] A. Colom, et al., Nature Communications, DOI:10.1038/ncomms3155 (2013) [6] F. Rico, et al., Science, 342, 741 (2013)

24

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Dissecting subunit assembly by single molecule imaging of membrane protein complexes Max Ulbrich [email protected]

Many ion channels and receptors exhibit a heteromeric composition with different subunit types assembled in a defined or random fashion. Biochemical methods have certain limitations that can make it difficult to determine the exact number and the stoichiometry of the subunits in a membrane protein. Single molecule techniques allow recording the properties of each individual protein complex, hereby preserving the full information about the ensemble and revealing subtle variations in complex composition, like a flexible stoichiometry or competition between certain subunit types. We have developed a single molecule imaging approach for counting subunits of membrane protein complexes in living cells. It is based on the direct observation of photobleaching steps from single fluorescent protein tags. We applied this technique to several important ion channels. By two color imaging we determine whether heteromeric channels form by random or directed assembly of their constituents.

25

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Novel trans-membrane mutation switches Orai1 to a constitutively active and Ca2+ selective channel Rainer Schindl, Irene Frischauf, Vasilina Zayats, Barbora Svobodova, Michael Deix, Martin Muik, Anna Hochreiter, Rüdiger Ettrich and Christoph Romanin Institute of Biophysics, Johannes Kepler University, Linz, Austria

The endoplasmic reticulum Ca2+ sensor STIM1 forms together with the Ca2+ channel Orai1 the molecular basis for Ca2+ release activated Ca2+ (CRAC) channels. The recent crystal structure of Orai from Drosophila melanogaster shows a unique Ca2+ channel composed of a hexameric subunit complex. The pore structure is formed by transmembrane (TM) 1 helices, surrounded by two ring-like structures, formed by TM2 and TM3 as well as TM4. Employing a combined approach of patch-clamp, molecular biology, biochemical techniques, molecular modeling and structure guided mutagenesis; we discovered a novel key mutation in the second trans-membrane helix of Orai1 that results in a Ca2+ selective, STIM1 independent, constitutively active current. Substitution of this essential residue to a hydrophobic amino-acid retained store-operated activation, yet with largely reduced Orai1 currents. In addition, we took advantage of the constitutively active Orai1 mutant, to evaluate reorientation of the gate located within the cytosolic region of TM1 helices. Cysteine scanning mutagenesis within the TM1 helix enabled identification of gating residues, the dimerization of which was altered in the constitutively opened and closed Orai1 channel conformation. Our experiments will be summarized in a unique gating model, and we will moreover discuss how STIM1 binding might trigger the open channel conformation. This work was supported by the Austrian Science Foundation (FWF): P26067 to R.S. and P25172 to C.R. Irene Frischauf is an Elise Richter Scholarship holder: V286.

26

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Kv channel activity determines the functional availability of smooth muscle BK channels in intact arteries Rudolf Schubert, Bettina Müller, Nadine Schmidt, Lena Devermann, Torsten Gloe, Dina Gaynullina, Mitko Mladenov Research Division Cardiovascular Physiology, Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, Heidelberg University, Germany

Introduction: Vascular smooth muscle high conductance calcium-activated potassium (BK) channels are activated by membrane depolarisation and an increase of the intracellular calcium concentration. These two factors also mediate constriction of blood vessels. Thus, vasoconstriction is accompanied by an activation of BK channels whereby the membrane hyperpolarisation produced by these channels limits the degree of constriction. Part of the activator calcium for the BK channel is provided by voltagegated calcium channels. The membrane potential of vascular smooth muscle cells is also under the control of voltage-gated potassium (Kv) channels. However, whether Kv channels, via their effect on the activity of voltage-gated calcium channels, are able to interact functionally with BK channels is unknown. Objectives: The hypothesis was tested that Kv channels functionally interact with BK channels in intact rat arteries. Materials & Methods: Experiments were performed on rat tail and saphenous arteries using realtime PCR for the analysis of BK and Kv channel expression, the patch-clamp technique for the direct measurement of potassium currents of intact cells and isometric myography to study vessel reactivity. Acute changes in vasoconstrictor tone were induced by the α1-receptor agonist methoxamine (MX). Results: MX produced a concentration-dependent contraction of tail and saphenous arteries. In the presence of the specific BK channel inhibitor iberiotoxin (IBTX) the MXinduced contractions were considerably increased, demonstrating the anticontractile effect of the BK channel. The latter effect was abolished completely after activation of Kv7 channels with retigabine and augmented considerably after blockade of Kv7 channels with XE991. Similar effects were observed in both vessels, despite the actions of retigabine and XE991 alone on MX-induced contractions being larger in the saphenous compared to the tail artery. Expression of BK channel α- and β1-subunits was similar in both vessels. However, while in the saphenous artery Kv7.1, Kv7.2 and Kv7.4 channel subunits were detected, in the tail artery only Kv7.4 channel subunits were found. In addition, BK current density was equal in both vessels, whereas Kv current density was larger in the saphenous compared to the tail artery. Conclusion: Our study demonstrates that in rat arteries BK channel control of vessel contractility is eliminated by Kv channel activation and enforced by Kv channel deactivation. This effect is independent on Kv channel availability. Thus, Kv channel activity determines the functional availability of smooth muscle BK channels in intact arteries.

27

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Voltage sensor conformational changes Francisco Bezanilla The University of Chicago

Voltage dependence of ion channels, transporters and some voltage-dependent enzymes is conferred by a voltage sensor (VS). The VS is normally a set of charges or dipoles embedded in the membrane protein that move in response to a change in the membrane potential. The movement of the charged voltage sensor produces gating (or sensing) currents which can be measured as transient currents in the external circuit under voltage clamp. Gating currents provide information of conformational states of the protein with a high level of temporal resolution and they have been the basis of detailed kinetic models that describe the gating of many channels, transporters and pumps. Although these electrical measurements are precise, they give no information on what parts of the structure are moving. Fluorescence spectroscopy provides spatial resolution with high sensitivity, to the single molecule level, and the measurements can be done while the protein is functional. Fluorescence spectroscopy is done after fluorescent probes are introduced in specific sites of the protein allowing the detection of time dependent quenching, spectral shifts, electrochromicity and energy transfer with simultaneous electrical measurement. In this presentation I will give examples of how fluorescence spectroscopy has provided detailed information on voltage sensing. Studies on Na+ and K+ channels and the voltage sensitive phosphatase Ci-VSP have provided a detailed picture of how a voltage sensor operates to reach different conformational states while revealing the molecular basis that makes Na+ channels faster than K+ channels, which is crucial for the generation of the nerve impulse. Support: NIHGM030376.

28

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Sunday, June 22nd, 2014 07.30 – 08.30

Breakfast

MODELLING AND THEORY OF PERMEATION 08.30 – 09.10 09.10 – 09.30

Jochen Hub (Göttingen) Energetics of Solutes at Membranes and Interfaces

Nicolas Wieder (Heidelberg) Fully Stochastic Modelling of the IP3 Receptor Channel

Stefan Howorka (London) 09.30 – 10.00

Membrane-Spanning DNA Nanopores: Biomimetic Chemical Structures for Single-Molecule Research and Nanotechnology

Petronel Tuluc (Innsbruck) 10.00 – 10.20

Residues critical for voltage-sensor transitions determining gating properties of CaV1.1

10.20 – 10.50

Coffee Break

THERMO- AND NOCICEPTIVE TRP CHANNELS Peter McNaughton (London) 10.50 – 11.30 11.30 – 12.10

Modulation of TRP channels - fundamental mechanisms and in vivo significance

Thomas Voets (Leuven) Dissecting and disturbing thermosensitive TRP channels

Katharina Held (Leuven) 12.10 – 12.30 12.30 – 13.00

A single agonist competent to open multiple ion permeation pathways in the nociceptor TRPM3

Marc Behrendt (Marburg) Differential regulation of TRPM3 splice variants

13.00 – 13.10

Closing

13.10 – 14.10

Lunch End of Meeting and Departure

29

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Energetics of Solutes at Membranes and Interfaces Florian Zocher1, Carl Caleman2, Maarten Wolf3, Gerrit Groenhof4, David van der Spoel5, Peter Pohl1, and Jochen S. Hub6,* 1

Johannes Kepler Universität, Institut für Biophysik, Linz, Austria Uppsala University, Department of Physics and Astronomy, Uppsala, Sweden 3 MPI for Biophysical Chemistry, Göttingen, Germany 4 University of Jyväskylä, Nanoscience Center, Finland 5 Uppsala University, Department of Cell and Molecular Biology, Uppsala, Sweden 6 Georg-August-University Göttingen, Institute for Microbiology and Genetics, Göttingen, Germany *Correspondence to [email protected] 2

Interfaces between water and hydrophobic media are prevalent in nature, including biological systems. We use atomistic molecular dynamics simulations to investigate the energetics of solutes at such interfaces. The first part of the talk focuses on the permeation of solutes across cholesterolcontaining membranes. In contrast to previous assumptions, the simulations suggest that variations in the partition coefficient, rather than in the diffusion constant, explain variations of membrane permeability by the action of cholesterol. We present results from recent scanning electrochemical microscopy that support these findings [1,2]. The second part of the talk focuses at ions at the water surface. It has long been debated whether the water surface is acidic or basic. Using new polarizable models, we derived the complete thermodynamics of hydronium and hydroxide at the water surface. The simulations suggest that hydronium, but not hydroxide, is slightly enriched at the water surface though an enthalpic effect [3]. Hydroxide is pushed into bulk water by entropy. The results are compared to the surface solvation of halide an alkali ions and discussed in the light of experimental data [4,5]. References [1] Florian Zocher, David van der Spoel, Peter Pohl, and Jochen S. Hub. 2014. Local partition coefficients govern solute permeability of cholesterol-containing membranes. Biophys. J. 105:2760-2770. [2] Christian Wennberg, David van der Spoel, and Jochen S. Hub. 2012. Large Influence of Cholesterol on Solute Partitioning into Lipid Membranes. J. Am. Chem. Soc. 134:5351-5361. [3] Jochen S. Hub, Maarten Wolf, Carl Caleman, Paul van Maaren, Gerrit Groenhof, and David van der Spoel. 2014. Thermodynamics of hydronium and hydroxide surface solvation. Chemical Science 5:1745-1749. [4] Jochen S. Hub, Carl Caleman, and David van der Spoel. 2012. Organic molecules on the surface of water droplets - an energetic perspective. PCCP, 14:9537-9545. [5] Carl Caleman, Jochen S. Hub, Paul J. van Maaren, and David van der Spoel. 2011. Atomistic simulation of ion solvation in water explains surface preference of halides. Proc. Natl. Acad. Scie USA 108:6838-3842.

30

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Fully Stochastic Modelling of the IP3 Receptor Channel Nicolas Wieder, Frederic von Wegner, Rainer Fink Universität Heidelberg, Institut für Physiologie und Pathophysiologie, AG Medizinische Biophysik, Heidelberg, Germany

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitous Ca2+ release channel that orchestrates the liberation of Ca2+ from the endoplasmic reticulum in a great variety of different cell types. Its biphasic non-linear regulation by Ca2+ and its structural organization in channel cluster are the basis of complex Ca2+ signal pattern such as waves and oscillations. Due to the low resting [Ca2+] of approximately 0.1 μM (~ 60 Ca2+ ions/fl) and the quantization of Ca2+ release sites in cluster microdomains, stochastic fluctuations are not negligible and classical deterministic simulation strategies reach their limitations. To explain the apparently random occurrences of Ca2+ puffs, elementary Ca2+ release events resulting from the synchronized opening of channel cluster, it becomes necessary to take the stochastic nature of underlying chemical reaction diffusion system into account. A great variety of stochastic modelling approaches exist, whereat the most fundamental formulation of exact stochastic reaction kinetics is the chemical master equation (CME) [1,2]. It describes the evolution of a system as a discrete time Markov process, based on reaction propensities that are closely related to macroscopic reaction rate constants. In contrast to stochastic differential equations where noise terms are usually arbitrary chosen, this approach reveals stochastic fluctuations with exact statistical properties. For complex systems, the CME quickly becomes analytical intractable and numerical simulations are the only option to gain information about the dynamic behaviour of the underlying system. Furthermore, due to the hierarchical spatial organization of Ca2+ signals ranging from the nm scale of single channels to the μm scale of global Ca2+ waves, the numerical solution of the CME becomes computational infeasible. One solution for these problems are hybrid algorithms that treat functionally important non-linear components (such as the IP3R) stochastically while passive bulk reactions and diffusion are treated deterministically [3]. Even though these approaches reveal promising results, they neglect intrinsic calcium signal noise with its system specific statistical properties, arising from dynamical sources (such as buffer association/dissociation reactions and diffusion). It is well known that noise can affect non-linear dynamics in biological systems [4] but only very little attention has been paid on the effects of Ca2+ signal noise on the gating dynamics of IP3Rs. We therefore use a fully stochastic description of a chemical reaction diffusion system to simulate an isolated IP3R in the vicinity of naturally fluctuating Ca2+ ions to examine the influence of Ca2+ signal noise on the non-linear gating dynamics of the IP3R. Our findings confirm the necessity of fully stochastic simulation strategies and reveal functional relevant aspects of Ca2+ signal noise. References [1] von Wegner, F., N. Wieder, and R. H. A. Fink, 2012. Simulation strategies for calcium microdomains and calcium-regulated calcium channels. Adv. Exp. Med. Biol. 740:553–567. [2] Wieder, N., R.H.A.Fink, and F.vonWegner, 2011. Exact and Approximate Stochastic Simulations of Intracellular Calcium Dynamics. J. Biomed. Biotechnol. 2011:1-5 [3] Rüdiger, S., J. W. Shuai, W. Huisinga, C. Nagaiah, G. Warnecke, I. Parker, and M. Falcke, 2007. Hybrid stochastic and deterministic simulations of calcium blips. Biophys. J. 93:1847–1857. [4] Longtin, A., 2003. Effects of noise on nonlinear dynamics. In Nonlinear Dynamics in Physiology and Medicine, Springer, 149–189.

31

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Membrane-Spanning DNA Nanopores: Biomimetic Chemical Structures for Single-Molecule Research and Nanotechnology Stefan Howorka University College London, Department of Chemistry, London WC1H0AJ, UK [email protected]

DNA nanotechnology excels at rationally designing bottom-up structures that can functionally replicate naturally occurring proteins. I describe the design and generation of stable self-assembled DNA-based nanopores that functionally mimic membrane protein pores and insert into lipid bilayers to support transmembrane water flow. The DNA nanopores consist of a bundle of six hexagonally arranged duplexes which are interconnected by cross-overs. The negatively charged nanobarrels carry lipid anchors to facilitate the pores’ insertion into the hydrophobic bilayers. The lipid anchors either neutralize localized negative charges on the DNA backbone to create a hydrophobic belt to resemble amphiphilic protein pores, as demonstrated with alkylated phosphorothioate groups (Nano Letters, 2013, 13, 2351). Alternatively, anchoring can be achieved with few, large hydrophobic group such as porphyrin which double as fluorophores (Angew Chem, doi anie.201305765). The nanoarchitectures are correctly assembled as confirmed by AFM, SEC, and DLS, and are fully functional as shown by single-channel current recordings. The small membrane-spanning DNA pores merge the fields of nanopores and DNA-nanotechnology and will help open up the design of entirely new molecular devices for applications within single-molecule research and sensing, electric circuits, catalysis, and nanofluidics.

32

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Residues critical for voltage-sensor transitions determining gating properties of CaV1.1 Petronel Tuluc*§, Vladimir Yarov-Yarovoy#, Bruno Benedetti* and Bernhard E. Flucher* *

Department of Physiology and Medical Physics, Medical University Innsbruck, Austria Department of Pharmacology and Toxicology, University of Innsbruck, Austria # Department of Physiology and Membrane Biology, UC Davies, USA §

Following membrane depolarization S4 segments of voltage-gated cation channels translocate through the membrane resulting in the opening of the channel pore. But, the translocation of the S4 positively charged amionoacids through the lipid bilayer is energetically unfavorable thus interactions with negatively charged aminoacids from S1 to S3 domains aid this process (1). The charge compensation mechanism has been extensively studied in potassium and sodium channels yet little is known about it in voltage-gated Ca2+ channels. Previously we have shown that the embryonic Cav1.1e splice variant, lacking exon 29 in the IVS3-S4 linker, has an 8-fold higher current amplitude and 30mV left-shifted voltage-sensitivity compared to the adult Cav1.1a splice variant (2, 3). Here we used ROSETTA modelling and site-directed mutagenesis to study the transition of the IVth voltage-sensing-domain (VSD) of CaV1.1 calcium channel splice variants and identify the role of S3-S4 loop in controlling S4 charge transfer during gating thus the Ca2+ channel voltage sensitivity. Modeling 4 consecutive states of the VSD suggested that the number of H-bonds formed between the R1 and R2 arginines of IVS4 and residues in IVS3 is different in Cav1.1e than in Cav1.1a. Among them aspartate at position 1196 (D1196) shows the strongest difference between the two splice variants. Indeed, voltage clamp recordings show that neutralizing the negative charge in S3 (D1196N) has no effect on the voltage sensitivity of Cav1.1a but confers poor voltage sensitivity to Cav1.1e. R1 and R2 neutralization in Cav1.1e shift the voltage dependence of CaV1.1e by at least 30 mV to the right while homologous mutations in CaV1.1a do not affect the voltage dependence. Together these findings suggest a model according to which the extracellular loop S3-S4 controls the orientation of S3 and S4 relative to each other. This alters charge transfer during gating and dramatically reduces the voltage sensitivity of CaV1.1a calcium channels. Support: FWF W1101, P23479, LFU-P7400-027-011 References 1. Tao, X., A. Lee, W. Limapichat, D. A. Dougherty, and R. MacKinnon. 2010. A gating charge transfer center in voltage sensors. Science 328:67-73. 2. Flucher, B. E., and P. Tuluc. 2011. A new L-type calcium channel isoform required for normal patterning of the developing neuromuscular junction. Channels (Austin) 5:518-524. 3. Tuluc, P., N. Molenda, B. Schlick, G. J. Obermair, B. E. Flucher, and K. Jurkat-Rott. 2009. A CaV1.1 Ca2+ channel splice variant with high conductance and voltage-sensitivity alters EC coupling in developing skeletal muscle. Biophysical journal 96:35-44.

33

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Modulation of TRP channels - fundamental mechanisms and in vivo significance Peter McNaughton Wolfson Centre for Age-Related Diseases, King’s College London

TRPV1 is an ion channel activated by painful levels of heat, while TRPM8 signals a sensation of pleasant coolness. The sensations imparted by activation of these channels are familiar to us from eating spicy food, which contains the selective TRPV1 agonist capsaicin, or mint, which contains menthol, a TRPM8 agonist. Both ion channels are important in pain sensation. Activation of TRPV1 contributes to the ongoing pain associated with injury, while TRPM8 has an analgesic action, as shown by the large number of over-thecounter analgesics which contain menthol. TRPV1 and TRPM8 might therefore be thought of as playing a yin-yang function in pain – activation of TRPV1 promotes pain, while activation of TRPM8 suppresses it. In recent work we have investigated how the function of TRPV1 and TRPM8 are modulated by inflammation. As might be expected, activation of TRPV1 is enhanced by inflammatory mediators such as prostaglandin E2, histamine or bradykinin, while activation of TRPM8 is suppressed. How is this achieved at a molecular level? Pro-inflammatory mediators enhance TRPV1 by activating kinases such as PKA and PKC, which in turn phosphorylate specific residues on TRPV1. We recently discovered that these two kinases depend for their action on a scaffolding protein, AKAP79, which assembles them into a signalling complex with TRPV1 (Zhang et al., 2008). If the interaction between AKAP79 and TRPV1 is prevented, for instance by infusing into the cell a peptide which competes for the binding site between them, then the effects of inflammatory mediators on TRPV1 are abolished. In more recent work (Btesh et al., 2013; Fischer et al., 2013) we have characterised the mutual binding sites on both TRPV1 and AKAP79, which we have localised to a short stretch of amino acid residues on each protein. Competitor peptides modelled on each site can be made cell-permeable, and in both cases these peptides have analgesic effects on inflammatory hyperalgesia in vivo. This binding site may therefore be an interesting future target for the development of novel analgesics. TRPM8 activation is suppressed by inflammation, but when we attempted to identify the critical signalling pathways it became clear that the mechanism must be different from that by which many inflammatory mediators enhance TRPV1. Agents which activate PKA or PKC had a potent effect in enhancing TRPV1 but had no effect on TRPM8; conversely inhibitors of classical signalling pathways did not reduce the effects of inflammatory mediators on TRPM8 (Zhang et al., 2012). We found that a much simpler signalling mechanism is responsible for inhibiting TRPM8: Gq, an important element of the G-protein signalling cascade, binds directly to and directly inhibits TRPM8 when activated by inflammatory mediators such as bradykinin or histamine. A direct action of Gq on an ion channel had not previously been demonstrated and adds to the repertoire of mechanisms by which G-protein coupled receptors signal to ion channels. References Btesh J, Fischer MJ, Stott K, McNaughton PA (2013) Mapping the Binding Site of TRPV1 on AKAP79: Implications for Inflammatory Hyperalgesia. J Neurosci 33:9184-9193. Fischer MJ, Btesh J, McNaughton PA (2013) Disrupting sensitization of transient receptor potential vanilloid subtype 1 inhibits inflammatory hyperalgesia. J Neurosci 33:7407-7414. Zhang X, Li L, McNaughton PA (2008) Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150. Neuron 59:450-461. Zhang X, Mak S, Li L, Parra A, Denlinger B, Belmonte C, McNaughton PA (2012) Direct inhibition of the cold-activated TRPM8 ion channel by Galpha(q). Nat Cell Biol 14:851-858.

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Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Dissecting and disturbing thermosensitive TRP channels Thomas Voets Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven

Temperature-sensitive TRP channels play a key role in somatosensory thermosensation. Most temperature-sensitive TRP channels are not only highly sensitive to changes in temperature but also to various natural and synthetic ligands. In this lecture, I will present recent data on the mechanisms whereby chemical ligands modulate the gating of temperature-sensitive TRP channels, illustrate how these ligands can be used to investigate the physiological function of these channels, and discuss the potential use of specific ligands for therapeutic purposes.

35

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

A single agonist competent to open multiple ion permeation pathways in the nociceptor TRPM3 Katharina Held1,2, Katrien De Clercq1, Hugo Klaassen3, Rieta Van Bree1, Barbara Colsoul2, Jean-Christophe Vanherck3, Patrick Chaltin3,4, Thomas Voets2 and Joris Vriens1 1

Laboratory of Obstetrics and Experimental Gynaecology, KU Leuven, Herestraat 49 box 611, B-3000 Leuven, Belgium 2 Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), KU Leuven, Herestraat 49 box 802, B-3000 Leuven, Belgium 3 Center for Drug Design and Development (CD3), Leuven, Belgium 4 CISTIM

TRPM3 is a calcium-permeable nonselective cation channel in the melastatin (TRPM) subfamily of TRP channels. It represents a typical example of a polymodally gated TRP channel, in that it can be activated by chemical ligands such as nifedipine and the neurosteroid pregnenolone sulphate (PS), as well as by physical stimuli such as heat and membrane depolarization. Recently, we have found evidence for an alternative ion permeation pathway distinct from the central pore, which can be gated by combined application of PS and exogenous chemicals such as clotrimazole. This alternative ion permeation pathway is preserved following desensitization, blockade, mutagenesis and chemical modification of the central pore, and enables massive Na+ influx at negative voltages. By screening a compound library, we identified CIM0216 as potent agonist of TRPM3. The compound exhibited a marked specificity for TRPM3 and induced [Ca2+]I signals in somatosensory neurons. Intriguingly, single application of CIM0216 was able to activate both the central pore ion permeation pathway and the alternative ion permeation pathway. Since physiological functions of TRPM3 and the alternative ion permeation pathway of TRPM3 are still poorly defined, the identification of a potent and selective activator is expected to contribute to clarifying the role of TRPM3 in vivo.

36

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Differential regulation of TRPM3 splice variants Marc Behrendt, Florian Mohr, Franziska Schneider, Sandeep Dembla and Johannes Oberwinkler* Philipps Universität Marburg, Institut für Physiologie und Pathophysiologie, Marburg, Germany *Correspondence to [email protected]

TRPM3 (transient receptor potential melastatin 3) proteins form calcium-permeable, nonselective cation channels that can be activated by the endogenous steroid pregnenolone sulfate [1]. These channels have been implicated in insulin release from pancreatic β cells [1] and in sensing noxious thermal stimuli in cutaneous nociceptor neurons [2]. However, it is still poorly understood how these channels are regulated by intracellular signaling cascades. Using electrophysiological techniques and calcium imaging we show that noradrenaline strongly inhibits TRPM3 activity via activation of α2-adrenoreceptors. Investigating the intracellular signaling cascades that mediate adrenergic TRPM3 inhibition we found that the mechanism is Gαi/o-protein coupled. However, direct action of Gαi/o-subunits on TRPM3 channels could be excluded through overexpression experiments. By contrast, overexpression of β/γ-subunits of heterotrimeric G-proteins strongly inhibited TRPM3 activity when both, β- and γ-subunits, were co-overexpressed. Additionally, co-immunoprecipitation experiments indicate that Gβ and TRPM3 may interact directly. Furthermore, we show that TRPM3 splice variants lacking 10 amino acids in the N-terminal part can neither be inhibited by α2-adrenoreceptor activation nor by μ-opioid receptor activation – both Gαi/o-protein coupled processes – providing evidence that Gβγ-mediated TRPM3 inhibition is dependent on highly specific protein-protein interactions. By mutational screening we characterize this putative Gβ-TRPM3 interaction site. Finally, we analyze the TRPM3 splice variant composition in distinct TRPM3 expressing cells. Together, our data indicate that TRPM3 channels are subject to intracellular regulatory mechanisms that allow fine-tuning the activity of these channels and the resulting calcium influx. References [1] Wagner, T. F., S. Loch, S. Lambert, I. Straub, S. Mannebach, I. Mathar, M. Düfer, A. Lis, V. Flockerzi, S.E. Philipp, and J. Oberwinkler. 2008. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells. Nat. Cell Biol. 10(12):1421-30. [2] Vriens, J., G. Owsianik, T. Hofmann, S. E. Philipp, J. Stab, X. Chen, M. Benoit, F. Xue, A. Janssens, S. Kerselaers, J. Oberwinkler, R. Vennekens, T. Gudermann, B. Nilius, and T. Voets. 2011. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 70(3):482-94.

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Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

POSTER ABSTRACTS Posters 1

2

Lucía Alonso-Carbajo (Leuven) Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells Sandeep Dembla (Marburg) Role of transient receptor potential melastatin 3 channels in the peripheral nervous system

3

Liudmila Erokhova (Linz) Water transport through the sodium-glucose cotransporter SGLT1

4

Christian Goecke (Marburg) Structural requirements for agonism at TRPM3 cation channels

5 6

Christian Halaszovich (Marburg) Engineered Voltage Sensing Phosphatases: What do they tell us about the gating mechanism? Andreas Horner (Linz) Mobility of water in a confined proteinaceous environment

7

Denis Knyazev (Linz) Voltage mediated gating of the bacterial protein translocation channel SecYEG

8

Lubica Lacinova (Bratislava) Gating of the CaV3.3 channel differs from gating of CaV3.1 and CaV3.3 channels

9

Andreas Lieb (Innsbruck) Molecular gating mechanism of CaV1.3 voltage gated calcium channels

10

Nadine J. Ortner (Innsbruck) Pyrimidine 2, 4, 6-triones are a new class of voltage-gated L-type Ca2+ channel activators

11

Elena Pohl (Wien) Impact of different aldehydes on the biophysical parameters of lipid bilayers reconstituted with UCP1

12

Alicia Sánchez Linde & Brett Boonen (Leuven) Cinnamaldehyde modulates capsaicin-evoked TRPV1 activation

13

Franziska Schneider (Marburg) TRPM1 channel activity is affected by mutations and G-protein coupled receptors

14

Azmat Sohail (Wien) Decrypting the Structure of LeuTAa Employing Luminescence Resonance Energy Transfer (LRET)

15

Balász I. Tóth (Leuven) Cellular regulation of transient receptor potential melastatin 3 (TRPM3) channel activity

16

Bettina Wilke (Marburg) A six Amino Acid Motif in the Proximal C-Terminus of TASK-3 Channels Conveys Diacylglycerol-mediated Inhibition

17

Lukas Winter (Linz) YidC forms a protein translocating channel 38

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells Lucía Alonso-Carbajo, Julio Alvarez-Collazo, Ana I. López-Medina, Yeranddy A. Alpizar, Sendoa Tajada, Bernd Nilius, Thomas Voets, José Ramón López-López, Karel Talavera, María Teresa Pérez-García and Julio L. Alvarez Cinnamaldehyde (CA), a major component of cinnamon, is an electrophilic compound that has been used in the study the mechanisms of pain and is also known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CAinduced activation of the chemosensory cation channel TRPA1 seems to be involved in these last phenomena, it has been shown that genetic ablation of TRPA1 is insufficient to abolish CA effects. In this study, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca2+ channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca2+ currents in mouse ventricular cardiomyocytes (VCM, with Ca2+ as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba2+ as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca2+ channels and that this effect may contribute to its vasorelaxing action. Importantly, these findings demonstrate that CA can not be identified as a potent specific agonist of the chemonociceptor TRPA1 and also indicate that the inhibition of voltage-gated Ca2+ channels should be taken into account when this compound is used to study the pathophysiological role of TRPA1.

39

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Role of transient receptor potential melastatin 3 channels in the peripheral nervous system Sandeep Dembla1, Marc Behrendt1, Florian Mohr1, Julia Stab2 and Johannes Oberwinkler1* 1

Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Marburg, Germany Department of Cell Biology and Applied Virology, Fraunhofer Institute for Biomedical Engineering, St.Ingbert, Germany *Correspondence to [email protected] 2

TRPM3 proteins belong to the large superfamily of TRP cation channels, are non-selective Ca2+ permeable channels, and are activated by the neurosteroid pregnenolone sulphate (1). TRPM3 channels are expressed in pancreatic β-cells (1) as well as in neurons of the dorsal root ganglia (2), where they act as mediators of insulin release or sensing noxious thermal stimuli in cutaneous nociceptor neurons, respectively. Ca2+ imaging and whole-cell patch clamp experiments have shown that a large subset of acutely isolated murine DRG neurons (up to 34%) are robustly activated if challenged with TRPM3 agonists (2). The activity of endogenous TRPM3 channels in neurons of dorsal root ganglia (DRG neurons) and their biological functions still remain poorly understood. Furthermore, how these channels are regulated by intracellular signaling cascades remains a big question. Employing Ca2+ imaging, we show that in HEK293 and in mouse primary DRG neurons, DAMGO inhibits TRPM3 activity via activation of µ-opioid receptors implicating an involvement of TRPM3 channels in analgesic pathways. Furthermore, naloxone, an antagonist of opioid receptors neither did inhibit nor did enhance TRPM3 activity. Experiments with pertussis toxin (PTX), an inhibitor of Gi/o-proteins, revealed that the mechanism is Gi/o-protein coupled in nociceptor neurons via µ-opioid receptor activation, which we have also seen in pancreatic β-cells via activation of α2-adrenoreceptors. In addition, noradrenaline also inhibited TRPM3 activity in mouse primary DRG neurons, but to a lesser extent than was seen in pancreatic β-cells. Fleetingly, we concentrate on the potential involvement of G-protein βγ-subunits to the opioid receptor inhibition of TRPM3 channels. Together, our data indicate that TRPM3 channels are subject to intracellular regulatory mechanisms that might potentially be involved in the opioid receptor mediated peripheral analgesia. References 1.

2.

Wagner, T. F., S. Loch, S. Lambert, I. Straub, S. Mannebach, I. Mathar, M. Dufer, A. Lis, V. Flockerzi, S. E. Philipp, and J. Oberwinkler. 2008. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells. Nat Cell Biol 10:1421-1430. Vriens, J., G. Owsianik, T. Hofmann, S. E. Philipp, J. Stab, X. Chen, M. Benoit, F. Xue, A. Janssens, S. Kerselaers, J. Oberwinkler, R. Vennekens, T. Gudermann, B. Nilius, and T. Voets. 2011. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 70:482-494.

40

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Water transport through the sodium-glucose cotransporter SGLT1 Liudmila Erokhova, Andreas Horner and Peter Pohl Johannes Kepler University, Institute for Biophysics, Linz, Austria Correspondence to [email protected]

The intestine reabsorbs about nine liters of water daily. A primary role in that water uptake has been ascribed to secondary active transporters that are thought to couple water transport to sodium transport along its concentration gradient. In contrast, homology structure-based molecular dynamics (MD) simulations suggest passive water movement through the most prominent member of the water transporting carrier family, the sodium-glucose cotransporter SGLT1. However, their calculated turnover numbers are three to four orders of magnitude higher than those determined experimentally. To clarify the water transporting mechanism of SGLT1, we used a previously established assay for determining the single molecule water permeability, p f, for transporters in epithelial cell monolayers (Erokhova et al., 2011). That is, we measured the dilution of an aqueous dye in the immediate vicinity of the monolayer by fluorescence correlation spectroscopy (FCS) and determined the abundance of the fluorescently labelled SGLT1 transporter in the apical and basolateral membranes by FCS. The pf value compares well to that reported for aquaporins. It indicates the presence of a passive water channel and is thus not compatible with secondary active water transport. We obtained independent proof by reconstituting the purified SGLT1 into lipid vesicles and observing shrinkage of these vesicles in an osmotic gradient. Channel abundance in the reconstituted membranes was determined as described previously (Knyazev et al., 2013), i.e. the number of vesicles in the confocal volume was determined by FCS and compared to that of SGLT1 containing micelles after having treated the vesicles with detergent. Mutational studies are under way to determine the water pathway. The project is supported by the Austrian Science Fund (P23574).

References: Erokhova, L., Horner, A., Kugler, P., and Pohl, P. (2011). Monitoring single-channel water permeability in polarized cells. J. Biol. Chem. 286, 39926-39932. Knyazev, D.G., Lents, A., Krause, E., Ollinger, N., Siligan, C., Papinski, D., Winter, L., Horner, A., and Pohl, P. (2013). The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem. 288, 17941-17946.

41

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Structural requirements for agonism at TRPM3 cation channels Christian Goecke, Marc Behrendt, Lana Schumann, Johannes Oberwinkler Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Marburg, Germany

TRPM3 channels are non-selective cation channels that have been implicated in a variety of functions, including the detection of noxious heat and the secretion of insulin. These channels can be activated by the endogenous steroid pregnenolone sulfate and the voltage-gated calcium channel blocker nifedipine, but it is unclear how these compounds interact with the channels. In electrophysiological and calcium imaging experiments it has been shown that pregnenolone sulfate and nifedipine bind to distinct binding sites. Furthermore, pregnenolone sulfate needs to bind to a chiral, and thus proteinaceous, binding site in order to activate TRPM3. Employing further structural analogs of pregnenolone sulfate, it has additionally been determined that the binding site of pregnenolone sulfate needs to accommodate a large, negatively charged substituent at the C3 position of the steroid backbone. By combining these data we devised a strategy to find candidate amino acid residues of TRPM3 important for channel activation. We systematically mutated positively charged amino acids accessible from the extracellular side (from which pregnenolone sulfate is capable of activating TRPM3). We then evaluated whether mutated channels that displayed a reduced response to pregnenolone sulfate still responded to nifedipine comparably to wild-type channels. We identified one mutant with these properties, which is predicted to be located at the transmembrane extracellular domain interface. Furthermore, we identified residues where amino acid substitution lead to a more pronounced decrease of the response to nifedipine. Our results support the idea that nifedipine and pregnenolone sulfate act through different intra-molecular pathways when activating TRPM3. These data will lead to a better understanding of the mechanisms of rapid chemical activation of TRPM3. Likely, this will help to identify more potent and specific pharmacological tools, with the ultimate goal to manipulate these channels for experimental and therapeutic purposes.

42

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Engineered Voltage Sensing Phosphatases: What do they tell us about the gating mechanism? Christian R. Halaszovich1, Michael G. Leitner, Angeliki Mavrantoni, and Dominik Oliver1,* 1

Philipps Universität Marburg, Institut für Physiologie, Abt. Neurophysiologie, Marburg, Germany

The discovery of voltage sensitive phosphatases (VSP) (1) brought a surprise addition to the family of voltage sensitive proteins, that up to this points contained only voltage gated ion channels. These enzymes are comprised of a four transmembrane segment voltage sensor domain (VSD) similar to the one commonly found in voltage gated ion channels and a phosphatase domain (PD), which is under the control of the VSD. How the VSD exerts this control is not well understood. Recent work by Hobiger et al. (2, 3) and Liu et al. (4) on Ciona intestinalis (Ci-) VSP proposed an interaction of positively charged amino acid residues in the linker, which connects the PD to the VSD, with negatively charged residues in the ‘TI’ / ‘gating’ loop of the PD, namely Asp400 and Glu402. In light of these findings we revisited our engineered VSPs PTENCiV (5) and hVSP1CiV (6), these being chimeras consisting of Ci-VSP’s VSD and the PD of PTEN or hVSP1, repectively. In those chimeras neither Asp400 or Glu402 (Ci-VSP numbering) are conserved. We found these chimeric enzymes to show robust voltage dependent phosphatase activity, similar to the one found in CiVSP. Mutations within the VSP-to-PD linker showed effects mirroring those previously reported for Ci-VSP, indicating an intact gating mechanism similar to the one found in wild-type Ci-VSP. Therefore we propose that the interactions described by Liu et al. (4) and Hobiger et al. (2, 3) are not a necessary prerequisite for the voltage dependent activation of VSPs. Further work will be required to establish the essential interactions between VSP, linker, and PD of VSPs. This work was supported by Deutsche Forschungsgemeinschaft (grant SFB 593, TP12 to D.O.), University Medical Center Giessen and Marburg (UKGM) (grant 32/2011MR to C.R.H.) References 1. 2. 3. 4. 5.

6.

Murata, Y., H. Iwasaki, M. Sasaki, K. Inaba, and Y. Okamura. 2005. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature 435:1239-1243. Hobiger, K., T. Utesch, M. A. Mroginski, and T. Friedrich. 2012. Coupling of Ci-VSP modules requires a combination of structure and electrostatics within the linker. Biophys J 102:1313-1322. Hobiger, K., T. Utesch, M. A. Mroginski, G. Seebohm, and T. Friedrich. 2013. The linker pivot in CiVSP: the key to unlock catalysis. PLoS One 8:e70272. Liu, L., S. C. Kohout, Q. Xu, S. Muller, C. R. Kimberlin, E. Y. Isacoff, and D. L. Minor, Jr. 2012. A glutamate switch controls voltage-sensitive phosphatase function. Nat Struct Mol Biol 19:633-641. Lacroix, J., C. R. Halaszovich, D. N. Schreiber, M. G. Leitner, F. Bezanilla, D. Oliver, and C. A. Villalba-Galea. 2011. Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential. J. Biol. Chem. 286:17945-17953. Halaszovich, C. R., M. G. Leitner, A. Mavrantoni, A. Le, L. Frezza, A. Feuer, D. N. Schreiber, C. A. Villalba-Galea, and D. Oliver. 2012. A human phospholipid phosphatase activated by a transmembrane control module. J. Lipid Res. 53:2266-2274.

43

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Mobility of water in a confined proteinaceous environment Andreas Horner,1,* Florian Zocher,1,* Johannes Preiner,2,* Nicole Ollinger,1 Christine Siligan,1 Sergey Akimov,3,4 Peter Pohl1,# 1

2

Johannes Kepler University Linz, Institute of Biophysics, Gruberstr. 40, 4020 Linz, Austria; Center for Advanced Bioanalysis GmbH (CBL), Gruberstr. 40, 4020 Linz, Austria 3 A. N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Leninskiy prospekt 31/4, Moscow 119071, Russian Federation 4 National University of Science and Technology ‘‘MISiS’’, Leninskiy prospekt 4, Moscow 119049, Russian Federation * Both authors contributed equally # Correspondence should be sent to [email protected]

Molecular dynamics simulations offer invaluable insight into protein folding and function. The respective contributions of water molecules e.g. in binding pockets, reactive centers, as constituents of proton conducting chains, as part of a hydrogen-bonded network in a certain conformation, or for channel gating are commonly calculated based on models which best reflect the properties of bulk water. However, the general applicability of these models is questionable. Here we show that water mobility inside proteins may exceed bulk water mobility by one order of magnitude. Our assessment of the intra-proteinaceous water diffusion coefficient is based on measurements of the unitary water channel permeability. We used stopped-flow experiments to determine the per-channel increment in proteoliposome water permeability as a function of protein abundance. Both fluorescence correlation spectroscopy and high speed atomic force microscopy served to determine the exact number of water channels per proteoliposome. The unitary water permeabilities of aquaporin-1, aquaporin-Z, and the glycerol facilitator GlpF exceeded previous estimates by one order of magnitude, indicating that the water diffusion constant inside these channels varies between 6.2x10-5 and 2.5x10-4 cm2s-1. The high mobility is consistent with previous reports on the low number of hydrogen bonds formed by the single file waters in the channel and the distorted geometry of these bonds.

44

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Voltage mediated gating of the bacterial protein translocation channel SecYEG Denis Knyazev, Christine Siligan, Lukas Winter, Peter Pohl Johannes Kepler University, Institute for Biophysics, Linz, Austria Correspondence to [email protected]

The engaged in protein transport bacterial translocon SecYEG must preserve the proton motif force during translocation. Though it holds for a resting state [1], SecYEG was shown to open as ion channel in response to ribosome binding [2]. In vivo and in vitro experiments with SecYEG mutants showed preservation of the barrier to small ions across the bacterial plasma membrane which was partly attributed to cation exclusion by the channel. Here we show that the purified and reconstituted SecYEG facilitates both cation and anion transport. But when engaged in translocation, the channel closes its gate to small molecules at physiological values of the membrane potential. This is also true for both plug deletion and pore ring mutants with a stalled translocation intermediate. The remaining leak current shows little ion selectivity and is two orders of magnitude smaller than the current through the open SecYEG channel. References: 1. Sapar M. Saparov, Karl Erlandson, Kurt Cannon, Julia Schaletzky, Sol Schulman, Tom A. Rapoport, and Peter Pohl (2007). Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell 26: 501-509. 2. Denis G. Knyazev, Alexander Lents, Eberhard Krause, Nicole Ollinger, Christine Siligan, Daniel Papinski, Lukas Winter, Andreas Horner, and Peter Pohl. The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem. 288 (25): 17941-17946.

45

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Gating of the CaV3.3 channel differs from gating of CaV3.1 and CaV3.3 channels Lubica Lacinova*, Maria Karmazinova Institute of Molecular Physiology and Genetics, Bratislava, Slovakia *Correspondence to [email protected]

CaV3 or low-voltage activated (LVA) calcium channels are distinguished by low voltage threshold for opening of the channel pore. Pore opening is preceded by an upward movement of S4 segments in domains I-IV, which are part of the channel’s voltage sensor. This movement could be measured as a so-called gating current (Qon). We compared gating currents of all three CaV3 channel subtypes. Channels were expressed in HEK 293 cells and whole-cell patch clamp was employed. Inward current was blocked by 30 μM of Er3+. CaV3 channels have virtually identical voltage-dependence of pore opening (conductance-voltage or G-V relation) but different kinetics of current activation with CaV3.3 being remarkably more slow than CaV3.1 and CaV3.2 [1]. In contrast to what is known for high-voltage activated calcium channels Qon-Vs of CaV3 channel’s gating are shifted towards more positive voltages than corresponding G-V relations and less than 40% of gating charge of LVA channels is transferred when G-V relations reach their plateau (Figure 1).

Figure 1. Normalized conductance-voltage (G/Gmax; open squares) relations for CaV3.1 (A), CaV3.2 (B) and CaV3.3 (C) relations are compared with voltage dependencies of on-gating charge (Qon/Qonmax; filled circles) for the same channel. Solid lines are Boltzmann fits of experimental data.

Positive shift between G-V and Qon-V was more pronounced in the CaV3.3 channel then in CaV3.1 and CaV3.2 channels. Kinetics of charge movement of CaV3.1 and CaV3.2 channels was virtually identical. Kinetics of charge movement of the CaV3.3 channel was almost 3-fold more slow. This is in line with more slow kinetics of pore opening. In conclusion, activation of voltage sensor of the Ca V3.3 requires higher depolarization of cell membrane and has slower kinetics compare to the Ca V3.1 and CaV3.2 channels despite of highly conserved amino acid sequence of S4 segments. Supported by VEGA 2/0044/13 References [1] Klockner, U., J. H. Lee, L. L. Cribbs, A. Daud, J. Hescheler, A. Pereverzev, E. Perez-Reyes, and T. Schneider. 1999. Comparison of the Ca2+ currents induced by expression of three cloned α1 subunits, α1G, α1H and α1I, of low-voltage-activated T-type Ca2+ channels. Eur J Neurosci 11:4171- 4178.

46

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Molecular gating mechanism of Cav1.3 voltage gated calcium channels Andreas Lieb, Alexandra Pinggera, Nadine J Ortner, Petronel Tuluc, and Jörg Striessnig* University of Innsbruck, Innsbruck, Austria. *Correspondence to [email protected]

Within the family of high voltage-gated Ca2+-channels (VGCC) Cav1.3 has unique negative threshold gating properties. These permit Cav1.3 to support sinoatrial pacemaking, hearing, and neuronal excitability. Recently Cav1.3 has also been linked to hypertension caused by aldosterone producing adenomas (1), and to excitotoxicity in the pathophysiology of Parkinson’s disease (2). The negative threshold of Cav1.3 Ca2+ currents can result from two distinct mechanisms: a more voltage sensitive voltage sensor movement (Qon) or an increased coupling efficiency (CE) of Qon to pore opening. Further fine-tuning of these biophysical properties are achieved by a C-terminal regulatory mechanism (CTM), which displaces calmodulin from its channel binding site. Alternative splicing can remove this CTM in short Cav1.3 splice variants and thereby increases Ca2+-dependent inactivation and enhances the voltage sensitivity of channel activation. Aim of this study was to determine if an increased CE, or a more negative Qon is responsible for this modulatory effect. We expressed Cav3.1 (a member of the low VGCC family), Cav1.3 and Cav1.2 α1-subunits (with α2δ1 and β3 subunits) in tsA-201 cells. Qon and inward calcium currents (ICa) were measured using the whole cell patch-clamp technique. We show that the more negative activation range of Cav1.3 current as compared to Cav1.2 is largely determined by a uniquely high voltage sensitivity of Q on. This activation of voltage sensors at negative potentials can even compensate for the low CE of Cav1.3, the lowest among the VGCC’s investigated in this study (3). However, splicing – induced removal of the CTM enhances CE but does not affect the voltage-dependence of QON. This facilitates opening of short Cav1.3 splice variants at negative voltages. We hypothesize that the C-terminus of long Cav1.3 splice variants interacts with intracellular domains that are involved in the transmission of Qon to the activation gate. References: 1.

2. 3.

Azizan, E. A., H. Poulsen, P. Tuluc, J. Zhou, M. V. Clausen, A. Lieb, C. Maniero, S. Garg, E. G. Bochukova, W. Zhao, L. H. Shaikh, C. A. Brighton, A. E. Teo, A. P. Davenport, T. Dekkers, B. Tops, B. Kusters, J. Ceral, G. S. Yeo, S. G. Neogi, I. McFarlane, N. Rosenfeld, F. Marass, J. Hadfield, W. Margas, K. Chaggar, M. Solar, J. Deinum, A. C. Dolphin, I. S. Farooqi, J. Striessnig, P. Nissen, and M. J. Brown. 2013. Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension. Nature genetics 45:1055-1060. Striessnig, J., A. Pinggera, G. Kaur, G. Bock, and P. Tuluc. 2014. L-type Ca channels in heart and brain. Wiley interdisciplinary reviews. Membrane transport and signaling 3:15-38. Lieb, A., N. Ortner, and J. Striessnig. 2014. C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to Pore Opening in Cav1.3 L-type Ca(2+) Channels. Biophys J 106:1467-1475.

47

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Pyrimidine 2, 4, 6-triones are a new class of voltage-gated L-type Ca2+ channel activators Nadine J. Ortner#1, Gabriella Bock#1, David H. F. Vandael2, Robert Mauersberger3, Henning J. Draheim4, Ronald Gust3, Emilio Carbone2, Petronel Tuluc1 and Jörg Striessnig1* 1

Department of Pharmacology and Toxicology, Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria 2 Department of Drug Science, Laboratory of Cellular and Molecular Neuroscience, Nanostructured Interfaces and Surfaces Center, 10125 Torino, Italy 3 Department of Pharmaceutical Chemistry, Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria 4 Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Birkendorfer Strasse 65, 88397 Biberach, Germany # equal contribution; * author for correspondence

In Parkinson’s disease (PD) L-type Ca2+ channel (LTCC) mediated dendritic Ca2+ oscillations during autonomous pacemaking may account for the specific cell loss of dopaminergic neurons in the substantia nigra pars compacta. Recent studies suggest that blocking these LTCC components may be neuroprotective in PD. LTCCs are characterized by their high sensitivity towards organic Ca2+ channel blockers. However, Cav1.2 and Cav1.3, the main brain LTCC isoforms cannot be discriminated efficiently in a pharmacological manner. Since inhibition of Cav1.2 could lead to blood pressure lowering side effects due to its high expression levels in the cardiovascular system, the development of Cav1.3-selective blockers is of great therapeutic interest. Cp8, a pyrimidine 2,4,6-trione derivative, has been recently claimed to be the first Cav1.3selective blocker. With Ca2+ (15 mM) or Ba2+ (10 or 15 mM) as charge carrier, the modulation of currents through Cav1.3 (rat or human long splice variant, rCav1.3L, hCav1.3L) and Cav1.2 (rabbit long or short C-terminus, rbCav1.2L, rbCav1.2S), transiently expressed with β3 and α2δ1 subunits in tsA-201 cells, was measured using the whole-cell patch-clamp technique. Unexpectedly, 50 μM Cp8 induced a change in gating kinetics closely resembling the activity of known LTCC activators such as FPL64176. This modulation was characterized by a slowing of activation and inactivation as well as an enhancement of tail currents. However, a minority of cells using Ba2+ as charge carrier showed no change in gating kinetics but a weak and non-selective inhibition of both channel isoforms. The activating properties of Cp8 could be further confirmed on native LTCCs in mouse chromaffin cells (MCCs; 2 mM Ca2+) where non-L-type currents were spared. Moreover, application of Cp8 also increased the spontaneous firing frequency of MCCs and the total Ca2+ load during an action potential. In conclusion, Cp8 induced a LTCC activator-like change in gating kinetics in all Ca2+ and the majority of Ba2+ recordings. A potent and Cav1.3-selective inhibition could not be observed. However, a weak and non-selective block of both, Cav1.2 and Cav1.3 Ba2+ currents, was seen in a minority of cells. Taken together, our data indicate that pyrimidine 2,4,6-triones can act as a new class of Ca2+ channel activators and reliable Cav1.3-selective agents still have to be discovered. This work was supported by the Austrian Science Fund (F44020, W11)

48

Permeation & Gating of Ion Channels

Strobl, June 20-22, 2014

Impact of different aldehydes on the biophysical parameters of lipid bilayers reconstituted with UCP1 Olga Jovanovic, Alina A. Pashkovskaya, Nadine Burchardt, Lars Gille, Elena E. Pohl Department of Biomedical Sciences, University of Veterinary Medicine, Vienna

E-mail: [email protected]

Oxidative stress generates a vast number of biologically active molecules, many of which might influence membrane protein function. In our previous work [1] we have shown that reactive aldehyde 4-hydroxy-2-nonenal (HNE) strongly potentiates the activation of uncoupling proteins (UCP) in the presence of free fatty acid (FFA), but is unable to activate UCP directly. Here we investigate the influence of other biologically important reactive aldehydes (RA) such as 4-oxo-2nonenal (ONE) and 4-hydroxy-2-hexenal (HHE) on proton transport function of UCPs and evaluate possible mechanisms. We test a hypothesis as to whether RAs influence lipid membrane parameters which lead to the facilitation of fatty acid transport e.g. membrane fluidity, conductance, dipole or surface membrane potentials. To compare the effects of RAs, we use artificial bilayer membranes of different lipid composition, reconstituted with UCP1 and FA [2]. We demonstrate that (i) all investigated RAs activate UCP1 but only in the presence of FAs, (ii) their activation potency increases in the range HHE