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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.

In order to specify the role of individual S4 segments in Ca V 1.2 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 Ca V 1.2 control the closed state in domain specific manner while stabilizing the open state in a non-specific manner., Competing Interests: The authors declare that they have no conflict of interest.

Voltage sensors (VSs) initiate the pore opening and closure in voltage-gated ion channels. Here, we propose a technique for estimation of the equilibrium constant of the up- and downward VS movements and rate constants of pore transitions from macroscopic current kinetics. Bell-shaped voltage dependence of the activation/deactivation time constants and Bolzmann distributions of CaV1.2 activation were analyzed in terms of a circular four-state (rest, activated, open, deactivated) channel model: both dependencies uniquely constrain the model parameters. Neutralization of gating charges in IS4 or IIS4 only slightly affects the equilibrium constant of VS transition while affecting simultaneously the rate constants of pore opening and closure. The application of our technique revealed that pore mutations on IS6-IVS6 segments induce pronounced shifts of the VS equilibrium between the resting (down) and activated (up) position. Analyzing a channelopathy mutation highlighted that the leftward shift of the activation curve induced by I781T on IIS6 is only partially (35 %) caused by a destabilization of the channel pore but predominantly (65 %) by a shifted VS equilibrium towards activation. The algorithm proposed for CaV1.2 may be applicable for calculating rate constants from macroscopic current kinetics in other voltage-gated ion channels.

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.

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 Ca(V)1.2. 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 (ΔV (act)). ΔV (act) 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 Ca(V)1.2.

The role of six cysteines of Ca(V)3.1 in channel gating was investigated. C241, C271, C282, C298, C313, and C323, located in the extracellular loop between segment IS5 and the pore helix, were each mutated to alanine; the resultant channels were expressed and studied by patch clamping in HEK293 cells. C298A and C313A conducted calcium currents, while the other mutants were not functional. C298A and C313A as well as double mutation C298/313A significantly reduced the amplitude of the calcium currents, shifted the activation curve in the depolarizing direction and slowed down channel inactivation. Redox agents dithiothreitol (DTT) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) shifted the current activation curve of wild-type channels in the hyperpolarizing direction. Activation curve for all mutated channels was shifted in hyperpolarizing direction by DTT while DTNB caused a depolarizing shift. Our study reveals that the cysteines we studied have an essential role in Ca(V)3.1 gating. We hypothesize that cysteines in the large extracellular loop of Ca(V)3.1 form bridges within the loop and/or neighboring channel segments that are essential for channel gating.

Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R-A-O and a deactivation O-D-R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A-O) and deactivation (D-R) are described by rate constants alpha and beta, and gamma and delta, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the "pore-releasing" sensor movement ((x(V)) was much weaker than the voltage dependence of "pore-locking" sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.

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.

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 Ca(v)1.2, 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 Ca(v)1.2 (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 Ca(v)1.2 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 Ca(v)1.2 is able to accommodate a large cation such as (-)D888.