Double Mutant Cycle Analysis Identified a Critical Leucine Residue in the IIS4S5 Linker for the Activation of the CaV2.3 Calcium Channel*

Mutations in distal S6 were shown to significantly alter the stability of the open state of CaV2.3 (Raybaud, A., Baspinar, E. E., Dionne, F., Dodier, Y., Sauvé, R., and Parent, L. (2007) J. Biol. Chem. 282, 27944–27952). By analogy with KV channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in CaV2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a −50 mV shift in the activation potential and a −20 mV shift in its charge-voltage relationship as compared with CaV2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔGinteract > 2 kcal mol−1) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔGinteract ≈6 kcal mol−1 suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated CaV channel.

Both in K V and Ca V channels, the question as to how the S4 motion is transmitted to the S6 helix to open the gate upon depolarization remains heavily studied. In the electromechanical coupling model based upon the K V 1.2 crystal structure (12), the S4S5 linker, which is located within atomic proximity (4 -5 Å) of the S6 helix, interacts with the latter in the closed state of the channel. The movement of the S4 voltage sensor is likely to induce a conformational change that pulls the S4S5 linker away from the S6 inner helix ultimately resulting in ions flowing through the pore. The closed conformation of the channel is postulated to be stabilized by hydrophobic interactions at the level of the "closed bundle" in S6 (13)(14)(15).
Experimental evidence supporting a role for the S4S5 linker in the voltage dependence of activation of K V channels is steadily mounting. Introduction of the S4S5 linker from KcsA disrupts the voltage-dependent activation of K V 2.1 (16,17). In hERG, cross-linking studies have shown that residues located in the S4S5 linker and in the S6 helix are in atomic proximity and that gating currents were greatly affected as a result of disulfide bridge formation (18). Furthermore, mutations of a unique glycine residue (Gly-546) within the N-terminal end of the S4S5 linker were shown to stabilize the closed state by ϳ1.6 -4.3 kcal/mol and restricted the voltage sensor movement (19).
The potential coupling mechanism between the S4S5 linker and the S6 helix has yet to be explored in Ca V channels. In contrast to K V channels, Ca V channels are structurally asymmetrical with four distinct albeit homologous S4S5 linkers and four distinct S6 helices. We have already shown that glycine substitutions in the distal S6 regions of domains I, II, III, and IV altered the relative stability between the open and closed states (20). Mutations within IIS6 were found to impact most significantly on the activation gating of Ca V 2.3. In particular, I701G shifted by Ϫ35 mV the voltage dependence of activation while slowing down inactivation and deactivation kinetics (20). These data indicated that IIS6 plays a unique role in the channel equilibrium between the closed and the open state(s) in Ca V 2.3.
To determine whether the S4S5 linker of Domain II (IIS4S5) is coupled with the S6 helix of the same domain (IIS6) during activation of Ca V 2.3, we performed a double mutant cycle analysis (21) within domain II of Ca V 2.3. Double mutant cycle analysis has often been proven to be a powerful tool for studying intra-protein interactions in Ca V 1.2 (22), K V (23,24), and CNG (25) channels among others. Our assay, conducted by introducing pairs of glycine residues, one in the IIS4S5 linker and one in the IIS6 helix, has identified crucial points of energy interaction between these two regions. Notably Leu-596 in IIS4S5 appeared to be strongly coupled with IIS6 distal residues from Leu-699 to Asp-704 with ⌬⌬G interact more negative than Ϫ2 kcal mol Ϫ1 . Altogether, our results provide the first series of evidence that the IIS4S5 and IIS6 regions are coupled during the activation of Ca V 2.3.
Functional Expression of Ca V 2.3-Oocytes were obtained from female Xenopus laevis clawed frogs as described previously (20,29). Oocytes were injected with 46 or 4.6 nl of a solution containing cRNA coding for the Ca V ␣1, Ca V ␣2b␦, and Ca V ␤3 subunits in a 6:2:3 weight ratio. Coexpression with the ancillary Ca V ␤3, which is predominant in the brain (30), promotes strong functional expression (31,32) and emphasizes closed-state inactivation (33) of Ca V 2.3. Functional expression of mutants was deemed significant with whole cell Ba 2ϩ currents larger than Ϫ300 nA.
Whole Cell Recording and Data Acquisition in Oocytes-Wild-type and mutant channels were screened at room temperature for macroscopic Ba 2ϩ currents, 2 to 4 days after RNA injection, using a two-electrode voltage-clamp amplifier (OC-725C, Warner Instruments) as described earlier (20,29,34). Oocytes were routinely injected with 23 nl of a 50 mM EGTA (Sigma) solution 0.5 to 2 h before the experiments. The pCLAMP 10 software (Molecular Devices, Sunnyvale, CA) was used for on-line data acquisition and analysis. A series of 500-ms voltage pulses (5 mV steps) was applied at a frequency of 0.2 Hz from a holding potential of Ϫ120 mV in most cases.   Biophysical parameters of Ca V 2.3 wild-type and mutant channels were estimated with Ca V ␣2b␦ and Ca V ␤3 in the presence of a 10 mM Ba 2ϩ solution as described elsewhere (29,38,41). The voltage dependence of inactivation was determined from the peak currents after 5-s depolarizing pulses from a holding potential of Ϫ120 mV (Ϫ140 mV for L596G). Activation properties (E 0.5,act and z) were estimated from the mean I-V relationships and fitted to a Boltzmann equation where z is the slope factor. The data are shown with the mean Ϯ S.E. of the individual experiments and the number of experiments appears in parentheses.

Ca
Ϫ4 Ϯ 2 (5) Ϫ0.5 Ϯ 0.  (35,36) with a CA-1B amplifier (Dagan Corp., Minneapolis, MN). The membrane of the oocyte exposed to the bottom chamber was permeabilized by a brief treatment with 0.1% saponin. The external solution contained 2 mM CoCl 2 , 110 mM NaOH, and 10 mM Hepes, titrated to pH 7.0 with methanesulfonic acid (37). The internal solution in contact with the oocyte cytoplasm was either 120 mM tetraethyl ammonium, 10 mM Hepes, 0.5 mM EGTA, and 5 mM MgSO 4 , titrated to pH 7.0 with methanesulfonic acid; or 110 mM potassium glutamate and 10 mM Hepes, titrated to pH 7.0 with KOH. Data acquisition and analysis were performed using the Digidata 1322A 16-bit system (Molecular Devices). Gating currents were filtered at 1 kHz and digitized at 10 kHz. Holding potential was Ϫ120 mV and 30-ms voltage steps were evoked from Ϫ100 to 60 mV by 5-mV increments. Linear components arising from charging the membrane and leak currents were eliminated by a P/Ϫ-4 protocol. Gating charge versus voltage (Q-V) relations were obtained by integrating the OFF gating currents and then fitting single Boltzmann functions (Equation 1) according to where parameter e is the elementary electric charge; k is the Boltzmann constant; z is the effective valence.
Data Analysis-Activation parameters were estimated from the peak I-V curves obtained for each channel combination and are reported as the mean of individual measurements Ϯ S.E. Briefly, the I-V relationships were normalized to the maximum amplitude and fitted to a Boltzmann equation with E 0,5,act being the midpotential of activation as described elsewhere (20,29,38). The estimation of E 0,5,act using nonstationary measurements rests upon the assumption that the transition to the open state is much faster than the transition to the inactivated state.
The free energy of activation was calculated using the midactivation potential by Equation 2.   Table 1. L596G yielded a significantly longer deactivation time constant (p Ͻ 0.001) than the Ca V 2.3 wt at all voltages more positive than Ϫ80 mV. D, representative tail currents for Ca V 2.3 wt and L596G. Currents were activated during 8-ms conditioning depolarizing voltage pulses at 0 mV for the wt and Ϫ40 mV for L596G. Deactivation was recorded during subsequent repolarization to test potentials from Ϫ120 to 0 mV (10-mV increments) as illustrated on the pulse protocol.
Deactivation time constants and inactivation parameters were calculated as described in detail under supplemental "Experimental Procedures". Statistical analyses were performed using the one-way analysis of variance fitting routine for two independent populations included in Origin 7.0. Data were considered statistically significant at p Ͻ 0.01.
Double Mutant Cycle Analysis-The coupling or interaction energy ⌬⌬G interact was calculated as, where ⌬G act,wt is the free energy of activation of the wild-type channel, ⌬G act,double is the free energy of activation of the double glycine mutant, ⌬G act,S4S5 is the free energy of activation of the single glycine mutant in the S4S5 linker, and ⌬G act,S6 is the free energy of activation of the single glycine mutant in S6.
⌬⌬G interact ϭ 0 suggests that the activation energies are purely additive hence that the residues are not functionally coupled. A value of ⌬⌬G interact more positive than ϩ1 kcal mol Ϫ1 or more negative than Ϫ1 kcal mol Ϫ1 (͉⌬⌬G interact ͉ Ͼ 1) was considered significantly different in similar studies (24,25). However, given the experimental variations associated with the measurements, we used a threshold of ͉⌬⌬G interact ͉ Ͼ 2 kcal mol Ϫ1 . Homology

RESULTS
Single Point Glycine Mutations in IIS4S5-A glycine scan was performed in the S4S5 linker of domain II to identify residues Activation gating properties of Ca V 2.3 wild-type and double mutant channels were estimated with Ca V ␣2b␦ and Ca V ␤3 in the presence of a 10 mM Ba 2ϩ solution as described elsewhere (29,38,41). Activation properties (E 0.5,act and z) were estimated from the mean I-V relationships and fitted to a Boltzmann equation where z is the slope factor. The data are shown with the mean Ϯ S.E. of the individual experiments and the number of experiments appears in parentheses. The biophysical properties of the L699G to A703G mutants were previously described (20) and are shown under "supplemental Table 1 Ϫ17 Ϯ 1 (5) a Ϫ1.9 Ϯ 0. most likely to play a role during the activation of Ca V 2.3. Because of the absence of a side chain, glycine mutations most likely minimize steric constraints, whereas generally decreasing the degree of hydrophobicity of the S4S5 linker. We used the predictive SAM-T02 algorithm (39) to evaluate the structural impact of introducing glycine residues in the S4S5 linker of domain II in Ca V 2.3 channels. As seen under supplemental Fig.  S1, single point mutations are not predicted to significantly alter the predicted secondary structure of this region. Glycine residues were thus introduced as single point mutations between residues 592 and 602 in the S4S5 linker of domain II (IIS4S5) (Fig. 1B). The ⌬G act and ⌬G inact (⌬G for the activation and inactivation, respectively) of these 11 consecutive single point mutations are reported in Table 1. As shown in Fig. 2, all mutant channels activated in response to membrane depolarization. From the histogram in Fig. 3A, it appears that the ⌬G act and ⌬G inact for most mutants (L592G, V593G, V594G, S595G, S598G, S599G, and K601G) were not significantly different from Ca V 2.3 wt. In contrast, the ⌬G act were significantly altered by glycine residues introduced at positions 596, 600, and 602 with a clear leftward shift in the voltage dependence of activation. It is noteworthy that no glycine mutants shifted ⌬G act toward positive voltages. L596G experienced the most significant change in the ⌬G act value from Ϫ1.0 Ϯ 0.1 kcal mol Ϫ1 (n ϭ 108) for Ca V 2.3 wt to Ϫ6.5 Ϯ 0.7 kcal mol Ϫ1 (n ϭ 6) for L596G yielding a ⌬⌬G act ϭ Ϫ5.5 Ϯ 0.8 kcal mol Ϫ1 . This singular effect was specific for the glycine mutation. Other nonconservative mutations at this position, L596D and L596P, were activated at significantly lower membrane potentials with ⌬G act ϭ Ϫ2.5 Ϯ 0.2 and Ϫ2.7 Ϯ 0.2 kcal mol Ϫ1 , respectively.
The voltage dependence of the Q/Q max curve for L596G was also significantly shifted toward negative voltages with V 0.5,Q ϭ Ϫ17 Ϯ 1 mV (n ϭ 4) as compared with V 0.5,Q ϭ 6 Ϯ 1 mV (n ϭ 19) for Ca V 2.3 wt (Fig. 3B). L596G displayed slower inactivation kinetics with a R50 ϭ 0.87 Ϯ 0.03 as compared with R50 ϭ 0.41 Ϯ 0.02 for Ca V 2.3 wt (Table 1), a significantly slower deactivation time constant (Fig. 3, C and D) Table S1 and Ref. 20) raising the possibility that the two regions contribute synergically to channel activation.
Leu-596 Is Coupled to Distal IIS6-To investigate the hypothesis that L596G in the S4S5 linker is specifically coupled to the distal S6 region of domain II, we performed a double mutant cycle analysis of these two regions. The double mutant cycle analysis provides a way for isolating the energetics of specific pairwise interactions (21,23). We opted to introduce pairs of glycine residues in IIS4S5 and IIS6, which had been shown to yield functional channels in our previous mutational analysis (20). The biophysical properties of a total of 39 double mutations produced between the distal IIS6 (699 to 704) (supplemental Table S1) and the 11 positions (592 to 602) in IIS4S5 were characterized from whole cell current recordings (supplemental Fig. S2).
Using Equation 3, ⌬⌬G interact were calculated for the double mutants with the ⌬G act and ⌬G inact values reported in Table 2 and supplemental Table S2. Fig. 4 shows the histogram of ⌬⌬G interact values for the activation of the 39 double mutants. The double mutant cycle analysis revealed cooperativity between L596G and 6 consecutive positions in the distal IIS6 (supplemental Fig. S3). Interaction was non-additive for the L596G double mutants with ⌬⌬G interact values ranging from 3 to 6 kcal mol Ϫ1 (see Table 2 for exact values). ⌬⌬G interact values larger than 1 kcal mol Ϫ1 are usually suggestive of a significant interaction (23,24) and exceed the coupling energies calculated between IS6 and IIS6 in Ca V 1.2 (22). Fig. 5 shows the whole cell FIGURE 4. Histogram of the interaction energies (⌬⌬G interact ) in kcal mol ؊1 for the activation of double mutants produced between IIS4S5 and IIS6 in Ca V 2.3. ⌬⌬G interact were calculated as described under "Experimental Procedures" using the ⌬G act values for the wt channel and the individual IIS4S5 mutants (see Table 1) as well as the ⌬G act values of the individual IIS6 mutants (see supplemental Table S1). Numerical values for the gating properties of the double mutants are shown in Table 2. In the histogram, the double mutants are shown with the IIS4S5 mutant to the left of the IIS6 mutant (going from the N-to the C-terminal). As shown, the pairs of residues V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, and S602G/I701G displayed significant coupling during the activation gating of Ca V 2.3. current traces, the average I/V curves, and the voltage dependence of the gating charge (Q/Q max ) of the L596G/I701G mutant. As seen, the gating charge curve for L596G/I701G was indistinguishable from either point mutations but significantly shifted to the left when compared with Ca V 2.3 wt. The absence of additivity in some the L596G/IIS6 mutants could thus arise in part from the dominant effect of L596G on the gating of Ca V 2.3. A similar observation can be made for I701G, the gating of which was not significantly modulated by any mutation in IIS4S5, save for M597G. The negative shift in the Q/V curve of I701G is reminiscent of the negative shift reported after mutation of the equivalent residue in S6 of Shaker K V channels (36). Altogether, these data suggest that Leu-596 and Ile-701 participate in a concerted fashion to the activation gating of Ca V 2.3.
Introducing a glycine residue at position 593 in IIS4S5 had little impact on the ⌬G act of Ca V 2.3. Nonetheless, V593G was significantly coupled with few distal IIS6 residues (L699G, A700G, A702G) during channel activation with ⌬⌬G interact values ranging from Ϫ2 to Ϫ3 kcal mol Ϫ1 (see Table 2 for exact values). V593G was not, however, coupled with I701G (supplemental Fig. S4). In addition to L596G, strong ⌬⌬G interact were measured with M597G (supplemental Fig. S5) and S602G during the transition from the closed to the open state.
Stabilizing the Open State Decreases Inactivation Kinetics-Preferential closed-state inactivation of Ca V 2.3 predicts that fully activated channels will be slower to inactivate than those in intermediate closed states (33). This prediction is tested in Fig. 6 where the inactivation kinetics are plotted against the ⌬G act of the L596G/IS6 and IIS4S5/I701G double mutants. As seen, double mutants with very negative ⌬G act values tend to inactivate more slowly (higher R 50 values). Coupling between S4S5 and S6 residues was also found to play a role in the closed state inactivation of K V 4.2 channels (40). Hence, altering the stability of the closed state relative to the stability of the open state influences inactivation kinetics. This contrasts with the minimal changes in the activation properties of the Ca V 2.3 mutants reported after mutations in the I-II linker (29,38,41,42). The ⌬⌬G interact values for the inactivation of the L596G double mutants (but not the V593G double mutants) were smaller than the ⌬⌬G interact values for activation ( Fig. 7 and supplemental Table S2).
Leu-589 Is Not Coupled with the Distal IIS6-The pivotal role played by a leucine residue in the gating of Ca V 2.3 is reminiscent of the situation in the Shaker channels where the heptad leucine motif in the S4S5 linker has been suggested to be important for its activation gating (43). The heptad motif being partially preserved between the S4S5 linker of Shaker channels and the IIS4S5 of Ca V 2.3, Leu-596 is predicted to be aligned with L3 in Shaker. In contrast, mutation of L2 to a valine (mutation V2) greatly destabilized the open state with a large positive shift in the activation potential (43). L2 of Shaker is predicted to be aligned with Leu-589 in Ca V 2.3. We investigated the gating properties of L589G and the double mutant L589G/I701G. In contrast to the observations in Shaker (43) and in hERG (19) channels, the activation gating of L589G was not significantly different from Ca V 2.3 wt (supplemental Table S3) and the double mutant L589G/I701G failed to display negative interaction energy.

DISCUSSION
In this study we have analyzed the role of the S4S5 linker of domain II in the activation gating of Ca V 2.3. Introducing glycine residues significantly affected ⌬G act when inserted at posi-  19) for Ca V 2.3 wt, Ϫ20 Ϯ 1 mV (n ϭ 15) for I701G, Ϫ24 Ϯ 1 mV (n ϭ 10) for L596G, and Ϫ22 Ϯ 1 mV (n ϭ 9) for L596G/I701G. tions 596, 600, and 602 with ⌬⌬G act ranging from Ϫ2 to Ϫ6 kcal mol Ϫ1 . The negative shifts of ⌬⌬G act observed with the glycine mutants are compatible with either a destabilization of the closed state or a stabilization of the open state, a distinction that requires single-channel recordings and goes beyond the scope of our current investigation.
Mutations in IIS4S5 brought changes in the activation potential of Ca V 2.3 that were comparable with those reported in homotetrameric K V channels (24) including mutations within the heptad leucine motif of Shaker channels (43). In the latter, mutations of L3 (aligned with Leu-596) to a valine (V3) or alanine (A3) residue shifted the midpotential for activation by Ϫ6 and Ϫ20 mV, respectively (43). In contrast, the alanine mutation of Leu-251, also predicted to be aligned with Leu-596, introduced a ϩ25 mV displacement in the activation potential of the KCNQ1 channel (24). Located in a more distal position, the E395A mutation (aligned with Ser-602), shifted by Ϫ20 mV the voltage dependence of the Q/Q max curve of Shaker channels (36).
None of the Ca V 2.3 single mutations herein studied were seen to completely uncouple the voltage sensor movement from pore openings, unlike I384N and F484G in homotetrameric Shaker channels (44). This may not be surprising given that our current investigation was limited to domain II. Despite the inherently asymmetrical nature of the Ca V 2.3 pore, it can only be surmised at this time that each domain contributes equally to the coupling between voltage sensor and channel opening as it has been demonstrated in Shaker channels (45).
A double mutant cycle analysis was carried out to identify pairs of IIS4S5 and IIS6 residues that could participate to the channel activation as done previously in K V channels (12,24,46,47). We had already shown that mutations carried out in the distal IIS6 impacted significantly on the relative stability of the closed state with negative shifts in the activation potential up to Ϫ40 mV (20). Our current analysis showed that Leu-596 in IIS4S5 and Ile-701 in IIS6 display significant energy coupling during channel activation. It is important to note that mutations of nearby residues in IIS4S5 displayed milder if any coupling with other residues in IIS6. The ⌬⌬G interact values measured between L596G and I701G were found to be stronger than the ones reported between conservative hydrophobic mutations performed in KCNQ1 (24) and CNG (25) channels. This interaction could be specific to Ca V 2.3. Double mutant FIGURE 6. A, correlation between the values of ⌬G act and R 50 for double mutations containing L596G. Values of R 50 for L596G double mutants were obtained as described under "Experimental Procedures" and are reported as a function of their associated ⌬G act . The linear regression was calculated with the routine included in Origin 7.0 and showed a strong negative correlation between these two values, with a R factor of Ϫ0.95. B, correlation between the values of ⌬G act and R 50 for double mutations containing I701G. Values of R 50 for I701G double mutants were obtained as described under "Experimental Procedures" and are reported as a function of their associated ⌬G act . The L596G/I701G mutant is indicated by a asterisk. The double mutant S598G/ I701G is shown but not identified on the graph. The linear regression was calculated with the routine included in Origin 7.0 and showed a strong negative correlation between these two values, with a R factor of Ϫ0.85.

FIGURE 7.
A, histogram of the ⌬⌬G interact for activation and inactivation of the V593G/IIS6 double mutants in Ca V 2.3. Residues Leu-699, Ala-700, and Ala-702 were significantly coupled with Val-593 during channel activation. As seen, ⌬⌬G interact for inactivation were not significantly different from ⌬⌬G interact for activation but for A702G. B, histogram of the ⌬⌬G interact for activation and inactivation of the L596G/IIS6 double mutants in Ca V 2.3. Residues Leu-699, Ala-700, Ile-701, Ala-702, Val-703, and Asp-704 were significantly coupled with Leu-596 during channel activation. As seen, ⌬⌬G interact for activation were stronger than ⌬⌬G interact for inactivation.
cycle experiments carried out in Ca V 1.2 and Ca V 2.1 with residues predicted to be aligned with Leu-596 and Ile-701 failed to show any significant energy coupling (supplemental Tables S4 and S5).
The molecular mechanism conveying Ca V 2.3 activation thus appears to rely in part on the electrochemical coupling between IIS4S5 and IIS6. Five of the six residues in IIS6 herein studied are hydrophobic suggesting that decreasing the hydrophobic content in either S4S5 or S6 might destabilize the closed state of the channel. Indeed, decreasing the hydrophobic content of IIS4S5 with L596D and L596P were also shown to alter the relative stability of the closed state in Ca V 2.3. The suggestion that the interaction between S4S5 and S6 is mostly hydrophobic (thus considered to be a weak interaction) is compatible with results obtained in Shaker-like (12,46) and KCNQ1 (24) channels and is further supported by the recent observation that S4S5 peptides inhibit KCNQ1 in a state-dependent manner (47).
The predictive locations of Leu-596 and Ile-701 are shown in the homology model of Ca V 2.3 based upon the atomic coordinates of K V 1.2 in the open state (Fig. 8). According to the model, it is plausible that Leu-596 and Ile-701 face each other in the protein. Nonetheless, the predicted distance of 10.6 Å between their C-␣ atoms (11.2 Å between their C-␤ atoms) makes a direct interaction unlikely. Leu-596 showed significant interaction with five other residues in distal IIS6 during the process that leads to Ca V 2.3 activation. This observation suggests that IIS6 may rotate outwardly, whereas the four S6 helices move away from one another. V593G, predicted to be facing away from Leu-596 in the computer-based model of the IIS4S5 linker, was also significantly coupled with a few positions in IIS6. Given the relative precision of the homology model, the possibility that Val-593 interacts with Ile-701 during channel activation cannot be completely ruled out. Cooperativity thus may reflect long range coupling of residues (21).
Nonetheless, many questions remain to be answered. Our study did not address whether the four voltage sensors act in a concerted or independent manner. It is thus impossible to argue that the S4S5/S6 coupling within domain II constitutes the obligatory rate-limiting voltage-dependent step for channel activation in Ca V 2.3. More importantly, it remains to be seen whether the cooperative mechanism in domain II requires atomic proximity between S4S5 and S6. These questions go beyond the scope of this current investigation and await further structural studies of Ca V channels.