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Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of S6 gates. Neutralization of S4 charges revealed that pore opening of CaV1.2 is triggered by a “gate releasing” movement of all four S4 segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Voltage clamp fluorometry, the cryo-EM structure of a mammalian calcium channel, biophysical and pharmacological studies, and mathematical simulations have all contributed to a novel interpretation of the role of voltage sensors in channel opening, closure, and inactivation. We illustrate the role of the different methodologies in gating studies and discuss the key molecular events leading CaV channels to open and to close. Electronic supplementary material The online version of this article (10.1007/s00424-018-2163-7) contains supplementary material, which is available to authorized users.

In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics. In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurally different S4 segments leave their resting positions not simultaneously. Upward movement of segments IS4 and (to a lesser extend) IIIS4 appear to be a rate-limiting stage for releasing the pore gates. These segments carry most of the effective charge for channel activation. Our study suggests that S4 segments of CaV1.2 control the closed state in domain specific manner while stabilizing the open state in a non-specific manner.

Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to −26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of the Boltzmann curve of inactivation was decreased from 17.4 ± 3.5 mV (IS4(K1Q)) to 6.2 ± 0.7 mV (IS4(K1Q/R2Q/R3Q/R4Q)). Neutralizations of single or multiple charges in IIS4 and IIIS4 did not significantly affect the time course of inactivation. Neutralization of individual IVS4 charges shifted the inactivation curve between 17.4 ± 1.7 mV (IVS4(R2Q)) and −4.6 ± 1.4 mV (IVS4(R4Q)) on the voltage axis and affected the slope of the inactivation curves (IVS4(R2Q): 10.2 ± 1.2 mV, IVS4(R4Q): 9.7 ± 0.7 mV and IVS4(K5Q): 8.1 ± 0.7 mV vs WT: 14.1 ± 0.8 mV). IS4(K1Q) attenuated while IS4(K1Q/R2Q/R3Q) and IS4(K1Q/R2Q/R4Q/R3Q) enhanced the development of inactivation. Shifts in the voltage dependence of inactivation curves induced by IS4 neutralizations significantly correlated with shifts of the voltage dependence of channel activation (r = 0.95, p

In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics. In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurally different S4 segments leave their resting positions not simultaneously. Upward movement of segments IS4 and (to a lesser extend) IIIS4 appear to be a rate-limiting stage for releasing the pore gates. These segments carry most of the effective charge for channel activation. Our study suggests that S4 segments of CaV1.2 control the closed state in domain specific manner while stabilizing the open state in a non-specific manner. Electronic supplementary material The online version of this article (doi:10.1007/s00424-016-1895-5) contains supplementary material, which is available to authorized users.

Blockade of rapid delayed rectifier current (IKr) can lead to cardiac arrhythmia, which is a major concern in drug discovery and development. Drug safety tests involve the voltage clamp measurements on HEK cells expressing hERG potassium channels (Kv11.1) and high affinity hERG channel inhibition is widely accepted predictor of Torsade de Point arrhythmias (TdP). There is, however, evidence that multiple ion channel block prevent EADs and TdP arrhythmias. To address this question we estimated the IC50 values of the established hERG channel inhibitor dofetilide and 13 derivatives using a planar patch clamp system and HEK cells expressing hERG (Kv11.1) channels. Additionally we examined action potential prolongation in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) in response to dofetilide and its derivatives. The arrhythmogenic potency was assessed by estimating the concentration required to prolong the action potential (AP) to 150% of control duration. In 11 out of 14 cases hERG inhibition was accompanied by a prolongation of AP the concentration of drugs inducing 150% increase of the AP duration correlated well with IC50 of hERG channels block (r = 0.94, p 200 µM). We conclude that the simultaneous block of depolarizing L-type calcium currents or cardiac sodium current offsets the effect of hERG block. Experimental data are supported by simulations of drug-induced changes in hiPSC-CMs action potentials. Our data support the new cardiac safety paradigm (CiPA) that discrimination between safe and unsafe drugs should address hERG inhibition and additionally their action on inward currents.

The Timothy syndrome mutations G402S and G406R abolish inactivation of Ca(V)1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of Ca(V)1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for Ca(V)1.2 activation and inactivation gating are discussed.

BACKGROUND AND PURPOSE Diltiazem inhibits CaV1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of CaV1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α1 subunits.

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2. Electronic supplementary material The online version of this article (doi:10.1007/s00424-010-0885-2) contains supplementary material, which is available to authorized users.

Voltage dependence and kinetics of Ca(V)1.2 activation are affected by structural changes in pore-lining S6 segments of the alpha(1)-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 ("bundle crossing region"), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471-38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 +/- 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 +/- 1.2 and L434T by -12.1 +/- 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 +/- 1.4 mV; L434T/I781T, -40.4 +/- 1.3 mV; L429T/L779T, -12.6 +/- 1.3 mV; and L434T/L779T, -22.4 +/- 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 +/- 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.

Background and purpose: Heterologous expression of a1, b2 and g2S(g1) subunits produces a mixed population of GABAA receptors containing a1b 2o ra1b2g2S(g1) subunits. GABA sensitivity (lower in receptors containing g 1o rg2S subunits) and the potentiation of GABA-activated chloride currents (IGABA) by benzodiazepines (BZDs) are dependent on g2S(g1) incorporation. A variable g subunit incorporation may affect the estimation of IGABA potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for mixed population of a1b2 and a1b2g2S(g1) receptors. Experimental approach: We investigated the relation between GABA sensitivity (EC50) and BZD modulation by analysing triazolam-, clotiazepam- and midazolam-induced potentiation of IGABA in Xenopus oocytes under two-microelectrode voltage clamp.

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Cav1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Cav1.2 (1). The shifted channel gating of these mutants (by 30–40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Cav1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Cav1.2 is able to accommodate a large cation such as (-)D888.

The hERG cavity can trap very bulky compounds, without perturbing normal gate closure., Inhibition of hERG K+ channels by structurally diverse drugs prolongs the ventricular action potential and increases the risk of torsade de pointes arrhythmias and sudden cardiac death. The capture of drugs behind closed channel gates, so-called drug trapping, is suggested to harbor an increased pro-arrhythmic risk. In this study, the trapping mechanisms of a trapped hERG blocker propafenone and a bulky derivative (MW: 647.24 g mol–1) were studied by making use of electrophysiological measurements in combination with molecular dynamics simulations. Our study suggests that the hERG cavity is able to accommodate very bulky compounds without disturbing gate closure.

GABA(A) receptors composed of alpha(1), beta(2), gamma(1) subunits are expressed in only a few areas of the brain and thus represent interesting drug targets. The pharmacological properties of this receptor subtype, however, are largely unknown. In the present study, we expressed alpha(1)beta(2)gamma(1)-GABA(A) receptors in Xenopus laevis oocytes and analyzed their modulation by 21 ligands from 12 structural classes making use of the two-microelectrode voltage-clamp method and a fast perfusion system. Modulation of GABA-induced chloride currents (I(GABA)) was studied at GABA concentrations eliciting 5 to 10% of the maximal response. Triazolam, clotiazepam, midazolam, 2-(4-methoxyphenyl)-2,3,5,6,7,8,9,10-octahydro-cyclohepta-(b)pyrazolo[4,3-d]pyridin-3-one (CGS 20625), 2-(4-chlorophenyl)-pyrazolo[4,3-c]quinolin-3-one (CGS 9896), diazepam, zolpidem, and bretazenil at 1 microM concentrations were able to significantly (>20%) enhance I(GABA) in alpha(1)beta(2)gamma(1) receptors. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, 3-methyl-6-[3-trifluoromethyl-phenyl]-1,2,4-triazolo[4,3-b]pyridazine (Cl 218,872), clobazam, flumazenil, 5-(6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)-3-methyl-[1,2,4]-oxadiazole (Ru 33203), 2-phenyl-4-(3-ethyl-piperidinyl)-quinoline (PK 9084), flurazepam, ethyl-7-methoxy-11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c] [1,4]benzodiazepine-1-carboxylate (l-655,708), 2-(6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)-4-methyl-thiazole (Ru 33356), and 6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)phenylmethanone (Ru 32698) (1 microM each) had no significant effect, and flunitrazepam and 2-phenyl-4-(4-ethyl-piperidinyl)-quinoline (PK 8165) inhibited I(GABA). The most potent compounds triazolam, clotiazepam, midazolam, and CGS 20625 were investigated in more detail on alpha(1)beta(2)gamma(1) and alpha(1)beta(2)gamma(2S) receptors. The potency and efficiency of these compounds for modulating I(GABA) was smaller for alpha(1)beta(2)gamma(1) than for alpha(1)beta(2)gamma(2S) receptors, and their effects on alpha(1)beta(2)gamma(1) could not be blocked by flumazenil. CGS 20625 displayed the highest efficiency by enhancing at 100 microM I(GABA) (alpha(1)beta(2)gamma(2)) by 775 +/- 17% versus 526 +/- 14% I(GABA) (alpha(1)beta(2)gamma(1)) and 157 +/- 17% I(GABA) (alpha(1)beta(2)) (p < 0.05). These data provide new insight into the pharmacological properties of GABA(A) receptors containing gamma(1) subunits and may aid in the design of specific ligands for this receptor subtype.

K(+) channels play a critical role in numerous physiological and pathophysiological processes rendering them an attractive target for therapeutic intervention. However, the hERG K(+) channel poses a special challenge in drug discovery, since block of this channel by a plethora of diverse chemical entities can lead to long QT syndrome and sudden death. Of particular interest is the so-called trapping phenomenon, characterized by capture of a drug behind closed channel gates, which harbors an increased pro-arrhythmic risk. In this study we investigated the influence of trapped blockers on the gating dynamics and probed the state dependence of dissociation in K(+) channels by making use of the quaternary tetrabutylammonium. By applying essential dynamics simulations and two-electrode voltage clamp we obtained detailed insights into the dynamics of trapping in KcsA and hERG. Our simulations suggest that the trapped TBA influences the F656 flexibility during gate closure. Based on these findings, we provide a structural hypothesis for drug trapping. Further our simulations reveal the extent of gate opening necessary for drug dissociation.

Understanding the molecular basis of hERG (Kv11.1) inhibition is essential for drug development. Here we systematically analyzed the mechanism and potency of hERG inhibition by a library of 13 dofetilide derivatives1. Currents through hERG channels stably expressed in HEK 293 cells were studied applying whole-cell configuration using planar patch clamp technique2.The estimated IC50s ranged between 4.3±0.7nM (LUF6200) and 297±77nM (LUF6139). An inverse relation was observed between potency (IC50) and rate of block development. Analysis of the kinetics of block development revealed that potency of dofetilide derivatives is predominantly determined by the drug dissociation rate constant. In general, higher molecular weight led to higher apparent drug affinity (r = 0.9).LUF6200, the most active compound, has 2 nitro groups in para position of the two aromatic rings and an ethyl moiety at the basic nitrogen atom. Pursuing a Topliss approach at the phenoxy group further supports the positive contribution of the nitro group1, showing that substituents with electron withdrawing groups in para position of the phenyl ring are beneficial for hERG blocking activity. This points towards a pi-pi interaction of at least one of the aromatic rings of dofetilide analogs with respective aromatic amino acids in the channel. Docking of the set of compounds into a homology model of the hERG channel supports this finding. A refined molecular model for dofetilide-hERG interaction based on the novel functional data and mutational studies is proposed.1. Shagufta et al. Exploring Chemical Substructures Essential for hERG K+ Channel Blockade by Synthesis and Biological Evaluation of Dofetilide Analogues. ChemMedChem 4, 1722-1732 (2009).2. Farre, C. & Fertig, N. HTS techniques for patch clamp-based ion channel screening - advances and economy. Expert Opin. Drug Discov. 7, 515-524 (2012).

Local anaesthetics (LA) exert their action by prolongation of the time course of repriming of voltage-gated Na channels after repolarization of the action potential. This may result from slow drug-dissociation from fast inactivated states or, alternatively, from stabilization of a native slow inactivated state (IM, Chen ZH et al. J Physiol. 524:37). The C-terminal part of transmembrane S6 segment of domain IV (DIV-S6) is an important determinant of inactivation gating and LA block. We performed serial cysteine scanning mutagenesis of positions 1575-1586 in DIV-S6 of rNav1.4 channels and investigated the relationship between changes in mutation-induced alterations of IM and respective changes in LA-induced prolongation of recovery. The constructs were expressed in TsA201 cells and studied by means of whole-cell patch-clamp technique. We examined the time course of recovery from fast and slow inactivation produced by 50 ms and by 10 s conditioning pulses to −20 mV, respectively. The LA Lidocaine (500 µM; LIDO) significantly slowed recovery from fast inactivation in all tested constructs except F1579C. The time constant of LIDO - induced slow recovery was ∼ 150 ms. Under LIDO-free conditions a similar time constant of slow recovery was found most constructs following 10 s depolarization, presumably reflecting recovery from native IM. However, the fraction of channels recovering from IM (F- IM) varied greatly among the tested constructs. LIDO significantly increased F- IM in most constructs. Interestingly, there was a significant negative correlation between F-IM without LIDO and the LIDO-induced increase in F- IM(R2 = 0.91; P < 0.0001). Furthermore, in M1585C F- IM was 0 under LIDO-free conditions but increased to 0.73±0.02 with LIDO. The data suggest that in the tested constructs slow recovery with LIDO reflects slow dissociation from fast inactivation rather than recovery from IM. Funding support: Austrian Science Fund W1232-B11.

Abstract— L-type calcium channels (Ca v 1.m) inactivate in response to elevation of intracellular Ca 2+ (Ca 2+ -dependent inactivation) and additionally by conformational changes induced by membrane depolarization (fast and slow voltage-dependent inactivation). Molecular determinants of inactivation play an essential role in channel inhibition by phenylalkylamines (PAAs). The relative impacts, however, of Ca 2+ -dependent and voltage-dependent inactivation in Ca v 1.2 sensitivity for PAAs remain unknown. In order to analyze the role of the different inactivation processes, we expressed Ca v 1.2 constructs composed of different β-subunits (β 1a -, β 2a -, or β 3 -subunit) in Xenopus oocytes and estimated their (-)gallopamil sensitivity by means of the two-microelectrode voltage clamp with either Ba 2+ or Ca 2+ as charge carrier. Ca v 1.2 consisting of the β 2a -subunit displayed the slowest inactivation and the lowest apparent sensitivity for the PAA (-)gallopamil. A significantly higher apparent (-)gallopamil-sensitivity with Ca 2+ as charge carrier was observed for all 3 β-subunit compositions. The kinetics of Ca 2+ -dependent inactivation and slow voltage-dependent inactivation were not affected by drug. The higher sensitivity of the Ca v 1.2 channels for (-)gallopamil with Ca 2+ as charge carrier results from slower recovery (τ rec,Ca ≈15 seconds versus τ rec,Ba ≈3 to 5 seconds) from a PAA-induced channel conformation. We propose a model where (-)gallopamil promotes a fast voltage-dependent component in Ca v 1.2 inactivation. The model reproduces the higher drug sensitivity in Ca 2+ as well as the lower sensitivity of slowly inactivating Ca v 1.2 composed of the β 2a -subunit.

The role of channel inactivation in the molecular mechanism of calcium (Ca 2+ ) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca 2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca 2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (I Ba ) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed I Ba recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of I Ba recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca 2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca 2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.

Human ether-a-go-go related gene (hERG) channel inhibitors can be trapped in the channels at rest. The structural peculiarities of hERG blockers that enable trapping or alternatively resting state dissociation are currently unknown. Propafenone (small molecule, MW 341 g/mol) is efficiently trapped in the closed hERG channel pore (1). To investigate whether the size of the blocking molecule plays a role in trapping we synthesized bulky propafenone derivatives containing benzoyl and trimethylphenyl side chains, attached by piperazine linkers, with molecular weights of 500 (Fba212) and 650 g/mol (Fba213) respectively. hERG channels were expressed in Xenopus laevis oocyte and potassium current inhibition was studied using the two - microelectrode voltage clamp technique.It was found: first, both compounds are potent hERG blockers with IC50 3.7μM (Fba212) and 52μM (Fba213). Secondly, channel block by Fba212 and 213 was prevented by mutations Y652A and F656A as previously shown for propafenone. Third, both propafenone derivatives were trapped at rest. To obtain insights into the molecular mechanism of channel block docking experiments with Fba212 and Fba213 in closed and open conformation were performed. Both compounds interact with the propafenone binding site (Y652A and F656A). Fba213 was found to exceed the size of the closed channel cavity of our hERG homology model. We conclude that drug trapping in hERG channels does not necessarily require full closure of the activation gate.1. Witchel HJ, Dempsey CE, Sessions RB, Perry M, Milnes JT, Hancox JC, Mitcheson JS.Mol Pharmacol. 2004 Nov; 66(5):1201-12.

Membrane depolarisation and upward movement of charged S4 segments initiates the opening of voltage gated calcium channels while repolarisation and S4 downward movement facilitates channel closure. The individual impact of segements IS4-IVS4 in CaV1.2 is largely unknown. CaV1.2 with a neutral IIS4 (IIS4N) activate and deactivate similar to wild type. A role of IIS4 in gating becames evident in CaV1.2 carrying positional specific mutations of glycines or alanines on one of the S6 gates (G432/A780/G1193/A1503, GAGA-mutations). In these slowly gating mutant channels neutralisation of IIS4N causes a paradoxical shift of the activation curve to the right, accelerates channel gating and thus partially rescues wild type gating.Here we make use of GAGA-S6-mutants to study the role of IS4 and IIS4 and the impact of individual S4 charges. Introduction of a single charge into neutralized IS4N (IS4N+R276) accelerated channel gating and reduced the the slope of the activation curve. IIS4N+R662, also accelerated gating and shifted the activation curve to more depolirized voltages but without significantly affecting its slope.Segments IS4 and IIS4 carrying either one or four charges at different positions all accelerated curent kinetics. Our data suggest different roles and impacts of IS4 and IIS4 in channel activation. IS4 appears to be more important for “unlocking” the closed resting gate while IIS4 contributes predominantly to stabilisation of the gate in the open conformation. A second finding was the different impact of partially and fully charged S4 segments on CaV1.2 gating. We hypothesise that the evolutionary design of CaV1.2 requires fife charges in IS4 and in IIS4 for voltage-dependent movements to their final up and down positions. Our data support a gating model of CaV1.2 where IS4 and IIS4 modulate all four cooperatively interacting S6 gates in a domain specific manner.

In continuation of our previous work (Stary et al. 2007, Beyl et al. 2010) we have analyzed the functional role of gating determinants in the bundle crossing region of Cav1.2. G432 (IS6), A780 (IIS6), G1193 (previous data, IIIS6) and A1503 (IVS6) were substituted with residues of different physicochemical properties. This ‘G/A/G/A’ motif was identified in a conservation analysis showing that Cav, Nav, TRP and many K channels contain glycines or alanines in equivalent positions. Most ‘G/A/G/A’ mutations shifted the activation curve in the hyperpolarizing direction, suggesting a destabilization of the closed and a stabilization of the open channel state. Mutations G432A/V, G1193A and A1503V did, however, stabilize the closed channel conformation as evident from rightward shifts of the activation curves. A comparison with the closed KcsA structure revealed that the ‘G/A/G/A’ motif (‘A/A/A/A’ in KcsA) is located at the narrowest point of the interface between two S6 (M2) helices, tightly packed in a hydrophobic environment. Our analysis suggests that size and hydrophobicity might be important for channel gating. We found that mutations to methionine or tryptophan either destabilize the closed channel or result in non-conducting channels. Small hydrophobic residues like alanine or valine stabilize the resting state. A comparison between resting and open state KcsA structures indicates that the ‘G/A/G/A’ motif and surrounding residues in Cav1.2 undergo large conformational changes upon gating, resulting in a displacement of more than 7 A.1 Stary, A., Shafrir, Y., Hering, S., Wolschann, P., Guy, H.R. (2007) Channels 2(3), 210-215.2 Beyl, S., Depil, K., Hohaus, A., Stary-Weinzinger, A., Timin, E., Shabbir, W., Kudrnac, M., Hering, S. (2010) Pflugers Arch, in press.

The inner cavity of the hERG potassium ion channel can accommodate large, structurally diverse compounds that can be trapped in the channel by closure of the activation gate. A small set of propafenone derivatives was synthesized, and both use-dependency and recovery from block were tested in order to gain insight into the behavior of these compounds with respect to trapping and non-trapping. Ligand-protein docking into homology models of the closed and open state of the hERG channel provides the first evidence for the molecular basis of drug trapping.

We have previously shown that mutations in pore lining segment IIS6 of Cav1.2 induce leftward shifts of the activation curve reflecting a destabilization of the closed and a simultaneous stabilization of the open channel state (Beyl et al. 2009). Systematic substitutions of a cluster of hydrophobic residues (LAIA motive) by residues of different size and polarity (except proline) revealed a strong correlation between changes in hydrophobicity (ΔH) and the shifts of the activation curve (ΔV0.5). A similar analysis in segment IS6 revealed no correlation between ΔH and ΔV0.5. Here we show that amino acid substitutions in the corresponding sequence stretch of segment IIIS6 (F1191 - I1196) shift the activation curves (ΔV0.5 F1191T=−7.1±1.3mV, V1192T= 8.2± 1.2mV, G1193T=-31.2±1.3mV, F1194T=2.1±1.2mV, V1195T=−10.3±1.1mV, I1196T=−17.9±1.1mV). A reduction in hydrophobicity in positions V1195 (ΔH(V1195T)=−4.9) and I1196 (ΔH(I1196T)=−5.2) apparently destabilized the closed channel state. Other mutations (V1192T, G1193T and F1194T) did not fit this paradigm. Potential interactions between residues in segments IS6, IIS6 and IIIS6 were analysed by means of double mutant cycle analysis. Our data reveal a positional specific interaction between gating determinants in segments IS6 and IIS6 as well as IIS6 and IIIS6. Rate constants of the voltage sensing machinery and pore stability were estimated using a 4 state gating model (Beyl et al. 2009).

Point mutations in a gating-related sequence stretch in the pore-lining segment IIS6 of CaV1.2 (779-782:LAIA motive) convert the high-voltage activated calcium channel into a low-voltage activated one. Here we analyze the changes in current activation and deactivation induced by these and glycine mutations in this region. Our model describes channel activation as voltage-dependent sensor movement and a voltage-independent pore opening and deactivation as voltage-dependent return of the sensor and subsequent pore closure.An inverse problem approach enabled the estimation of current activation and deactivation rate constants from 16 mutants and wild type CaV1.2 with narrow confidence intervals. Current activation, deactivation and steady-state activation of wild type and 12 mutants could be fitted with identical voltage dependencies of the voltage sensing machinery (x(V),y(V)). Steeper voltage dependence of y(V) compared to x(V) (see Figure) suggest that a membrane hyperpolarisation more efficiently closes than a depolarization opens the channel. Mutations in IIS6 of CaV1.2 destabilizing the closed state simultaneously appear to stabilize the open state in all 16 mutants.∗ This study was supported by a grant from FWF (Project P19614-B11).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Calcium channel family members activate at different membrane potentials, which enables tissue specific calcium entry. Pore mutations affecting this voltage dependence are associated with channelopathies. In this review we analyze the link between voltage sensitivity and corresponding kinetic phenotypes of calcium channel activation. Systematic changes in hydrophobicity in the lower third of S6 segments gradually shift the activation curve thereby determining the voltage sensitivity. Homology modeling suggests that hydrophobic residues that are located in all four S6 segments close to the inner channel mouth might form adhesion points stabilizing the closed gate. Simulation studies support a scenario where voltage sensors and the pore are essentially independent structural units. We speculate that evolution designed the voltage sensing machinery as robust “all-or-non” device while the varietys of voltage sensitivities of different channel types was accomplished by shaping pore stability.

Valerian is a commonly used herbal medicinal product for the treatment of anxiety and insomnia. Here we report the stimulation of chloride currents through GABA(A) receptors (I(GABA)) by valerenic acid (VA), a constituent of Valerian. To analyse the molecular basis of VA action, we expressed GABA(A) receptors with 13 different subunit compositions in Xenopus oocytes and measured I(GABA) using the two-microelectrode voltage-clamp technique. We report a subtype-dependent stimulation of I(GABA) by VA. Only channels incorporating beta(2) or beta(3) subunits were stimulated by VA. Replacing beta(2/3) by beta(1) drastically reduced the sensitivity of the resulting GABA(A) channels. The stimulatory effect of VA on alpha(1)beta(2) receptors was substantially reduced by the point mutation beta(2N265S) (known to inhibit loreclezole action). Mutating the corresponding residue of beta(1) (beta(1S290N)) induced VA sensitivity in alpha(1)beta(1S290N) comparable to alpha(1)beta(2) receptors. Modulation of I(GABA) was not significantly dependent on incorporation of alpha(1), alpha(2), alpha(3) or alpha(5) subunits. VA displayed a significantly lower efficiency on channels incorporating alpha(4) subunits. I(GABA) modulation by VA was not gamma subunit dependent and not inhibited by flumazenil (1 microM). VA shifted the GABA concentration-effect curve towards lower GABA concentrations and elicited substantial currents through GABA(A) channels at > or = 30 microM. At higher concentrations (> or = 100 microM), VA and acetoxy-VA inhibit I(GABA). A possible open channel block mechanism is discussed. In summary, VA was identified as a subunit specific allosteric modulator of GABA(A) receptors that is likely to interact with the loreclezole binding pocket.

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. Here, we use molecular dynamics (MD) simulations with an applied membrane potential to investigate the ion flux of bacterial sodium channel NaVMs. 5.9 µs simulations with 500 mM NaCl suggest different mechanisms for inward and outward flux. The predicted inward conductance rate of ∼27±3 pS, agrees with experiment. The estimated outward conductance rate is 15±3 pS, which is considerably lower. Comparing inward and outward flux, the mean ion dwell time in the selectivity filter (SF) is prolonged from 13.5±0.6 ns to 20.1±1.1 ns. Analysis of the Na+ distribution revealed distinct patterns for influx and efflux events. In 32.0±5.9% of the simulation time, the E53 side chains adopted a flipped conformation during outward conduction, whereas this conformational change was rarely observed (2.7±0.5%) during influx. Further, simulations with dihedral restraints revealed that influx is less affected by the E53 conformational flexibility. In contrast, during outward conduction, our simulations indicate that the flipped E53 conformation provides direct coordination for Na+. The free energy profile (potential of mean force calculations) indicates that this conformational change lowers the putative barriers between sites SCEN and SHFS during outward conduction. We hypothesize that during an action potential, the increased Na+ outward transition propensities at depolarizing potentials might increase the probability of E53 conformational changes in the SF. Subsequently, this might be a first step towards initiating slow inactivation., Author Summary Voltage gated sodium channels are essential components of living cell membranes. They regulate the cell potential by facilitating permeation of ions across the membrane. In the past decades, studies revealed that the bacterial selectivity filter (SF) exhibits a constricted architecture lined with electronegative carboxyl oxygens of four glutamic acid side chains (EEEE motif), which repulse anions but attract Na+ ions. Crystal structures enable the investigation of structural dynamics with computational methods. Ion selectivity and conduction mechanisms between Na+, K+ and Ca2+ are progressively elucidated by molecular dynamics simulations and free energy calculations. The structural dynamics of the protein, especially the flexibility of SF and its fundamental role in kinetics underpinning ion selectivity, conduction and channel gating are less well understood. To shed light on this question, we use computational simulations to simulate ion conduction with membrane potentials. Our results suggest different dynamical behaviors of the EEEE locus and distinct ion distribution patterns in the SF with respect to permeating directionalities. These findings indicate a novel mechanism in differentiating reciprocal transitions of ion flow, preventing large sodium efflux during action potential initiation and may further suggest that increased flipping propensities at depolarizing potentials, might initially trigger channel slow inactivation.

Unexpectedly, thetransfer of the key amino acidsinvolved in sensitivity to DHPs,PAAs and BTZs results not only inthe transfer of the high-affinity drug-binding sites but also in the transferof a whole complex of propertiesthat characterizes the interaction ofthe drug with the native, donor L-type channels

Voltage sensors initiate the closed - open transitions in the ion conducting pore. Here we consecutively neutralized the charges of the four S4 segments (IS4-IVS4) of CaV1.2. The channel construct lacking charges in IIS4 (IIS4N) conducted barium currents. Surprisingly, charge neutralization accelerated channel closure and diminished shifts of the activation curves also in S6 mutants (IS6: G432W/IIS4N, IIS6: A780T/IIS4N, IIIS6: G1193T/IIS4N, IVS6: A1503G/IIS4N). Such a rescue of S6-mediated gating perturbations is exemplified for the IVS6 mutation A1503G. A1503G induced a −22.6±1.2 mV shift of the activation curve to the left that was diminished by charge neutralization in IIS4. Gating disturbances induced by pore mutations upstream and downstream of G432, G1193 and A1503 were unaffected. Thermodynamic analysis reveals that segment IIS4 interacts (energetically coupled) with a ring of small homologous amino acids G432, A780, G1193 and A1503 (G/A/G/A ring, Depil et al. 2011) in all four pore forming S6 segments.The research was funded by the Austrian Science Fund (FWF): grant P22600-B12 and P19614.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Diltiazem inhibits CaV1.2 channels and is widely used in clinical practiceto treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of CaV1.2. Here we elucidate the access path of Diltiazem to its binding site making use of membrane impermeable quaternary derivative qDil and mutant α1 subunits. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion). qDil inhibits CaV1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59±4% at 300 µM) and induced only a low level of tonic (non use-dependent) block (16±2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 reduced sensitivity to intracellularly applied qDil.Our study demonstrates intracellular access of quaternary diltiazem and its interaction with previously identified determinants of BTZ binding pocket. Recovery from block by diltiazem was found to be voltage independent, which is in contrast with the pronounced voltage dependent recovery from block by phenylalkylamines.

It was suggested about 20 years ago that drugs can bind with different strengths to resting (R), open (0), and inactivated (I) conformational states of ionic channels (Strichartz, 1973; Courtney, 1975; Khodorov et al., 1976; Hille, 1977; Hondeghem and Katzung, 1977). In the 1980s this concept was applied to the mechanism of action of calcium channel ligands on L-type calcium channels in heart (Bean, 1984; Sanguinetti and Kass, 1984; McDonald et al., 1984) and smooth muscle cells (Bean et al., 1986; Klockner and Isenberg, 1986; Hering et al., 1988). Patch-clamp studies in single cells enabled an accurate description of drug-induced changes in whole-cell current kinetics and single-channel behavior and subsequently the interpretation of electrophysiological data in terms of channel state models. Comparative studies of the action of 1,4dihydropyridine (1,4-DHP), phenylalkylamine (PAA), and benzothiazepine (BTA) in single cardiomyocytes revealed remarkable differences with respect to the dependence of drug action on the frequency of depolarization and membrane potential (Lee and Tsien, 1983; Uehra and Hume, 1985). In analogy to the effect of local anesthetics on sodium channels (Khodorov, 1981), the action of antagonist 1,4-DHPs has been interpreted as a high-affinity drug binding to inactivated calcium channels (Bean, 1984; Sanguinetti and Kass, 1984). The action of 1,4-DHPs, however, can be distinguished from “lidocain-like” drugs, as the 1,4-DHP effect on calcium channels is not significantly enhanced by repetitive membrane depolarization (Lee and Tsien, 1983; Uhera and Hume, 1985; Hering et al., 1988). A strong dependence on the “use of calcium channels,” i.e. activation of channels by test pulses, is evident for PAA antagonists. Their action seems to be closely related to the open conformational state (McDonald et al., 1984; Oyama et al., 1987; Hering et al., 1989).

Inhibition of hERG channels prolongs the ventricular action potential and correspondingly the QT-interval with the risk of torsade de pointes arrhythmias which may result in sudden cardiac death.[...]

Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of Ca(V)1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4(N)), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4(N) channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments ("GAGA ring": G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4(N) was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6-IVS6, which also affect activation gating, were not rescued by IIS4(N). Thus, the rescue of gating distortions in segments IS6-IVS6 by IIS4(N) is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.

A channelopathy mutation in segment IIS6 of Ca(V)1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca(V)1.2 (midpoint of activation curve (V(0.5)) shifted to -37.7 +/- 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V(0.5) of I781T/N785A (V(0.5) = -21.5 +/- 0.6 mV) was shifted back towards wild-type (V(0.5) = -9.9 +/- 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca(V)1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.

Background Heterologous expression of α1, β2 and γ2S(γ1) subunits produces a mixed population of GABAA receptors containing α1β2 or α1β2γ2S(γ1) subunits. GABA sensitivity (lower in receptors containing γ1 or γ2S subunits) and the potentiation of GABA-activated chloride currents (IGABA) by benzodiazepines (BZDs) are dependent on γ2S(γ1) incorporation [1]. A variable γ subunit incorporation may affect the estimation of IGABA potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for a mixed population of α1β2 and α1β2γ2S(γ1) receptors.

Results Homology modeling revealed that G432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (G432 (IS6), A780 (IIS6), G1193 (IIIS6), A1503 (IVS6)). In contrast, corresponding mutations in domains II, III and IV induced parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of G432 in domain I. Mutations of G432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices.