The Timothy syndrome mutations G402S and G406R abolish inactivation of CaV1.2 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 CaV1.2 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 CaV1.2 activation and inactivation gating are discussed.

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 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 materialThe online version of this article (doi:10.1007/s00424-010-0885-2) contains supplementary material, which is available to authorized users.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1319-8) contains supplementary material, which is available to authorized users.

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