Asymmetric contribution of a selectivity filter gate in triggering inactivation of CaV1.3 channels

Figure 1.. Asymmetric role of DIV pore helix in tuning VDI of CaV1.3. (A) Three distinct end-stage mechanisms for CaV1.3 inactivation. Scheme 1 shows a hinged-lid mechanism where the pore is occluded by an inactivation particle (DI–DII linker), scheme 2 shows an allosteric mechanism where the open probability is reduced through increased channel closure, and scheme 3 shows selectivity filter collapse. (B) Sequence alignment shows the conservation of a tryptophan residue (W) in the pore segment adjacent to the putative Ca2+ coordinating glutamate (E) residue. (C) Structural models show the pseudo-tetrameric architecture of CaV1.3 (PDB ID 7UHG). The inset shows the ion conduction pathway lined by the selectivity filter Ca2+ coordinating glutamate residues and the spatial location of the conserved tryptophan. (D) Representative Ba2+ current traces evoked in response to an 800-ms voltage step to 0 mV show minimal VDI of both wild-type (gray) and DI W[+2]A mutant channels. The wild-type current is normalized to compare inactivation kinetics. (E and F) Similarly, both DII W[+2]A and DIII W[+2]A mutant channels also showed minimal VDI. Format as in panel C. (G) By contrast, DIV W[+2]A mutation…

Figure S1.. CaV1.3 DI–DIV SF mutations minimally perturb channel activation. (A) Normalized current–voltage relationship for WT CaV1.3. Each dot, mean ± SEM n = 5 cells. (B–E) Normalized IV relations for DI W[+2]A (panel B, n = 5 cells), DII W[+2]A (panel C, n = 6 cells), DIII W[+2]A (panel D, n = 5 cells), and DIV W[+2]A (panel E, n = 7 cells) mutants. In all cases, the change in channel half-activation potential (V1/2) was <5 mV.

Figure 2.. Distinct functional signature of CaV1.3 VDI. (A and B) Exemplar Ba2+ current traces evoked in response to a family of voltage-steps show VDI of both wild-type CaV1.3 (panel A) and DIV W[+2]A mutant (panel B). CaM1234 coexpressed to exclude the potential contribution of CDI. (C) A two-pulse protocol is used to quantify the voltage dependence of inactivation. Comparison of Ba2+ currents for wild-type CaV1.3 during the prepulse versus the postpulse. CaV1.3 currents are increased in response to higher test-pulse potentials. See Fig. S2 B for full exemplar trace. (D) DIV W[+2]A mutant channels exhibit diminished peak currents during the postpulse following test pulse to intermediate voltages. See Fig. S2 C for a full exemplar trace. (E) Analysis of wild-type CaV1.3 reveals minimal VDI across multiple voltages. For each dot and error, mean ± SEM n = 7. (F) VDI of DIV W[+2]A mutation exhibits a U-shaped voltage-dependence with the strongest inactivation observed near 0 mV. Blue curve, voltage-dependence of inactivation obtained from the whole cell normalized IV relations (Fig. S2 E). For each dot and error, mean ± SEM n = 7 cells. (G) Exemplar traces show similar kinetics of…

Figure S2.. Extended biophysical characterization of DIV W[+2]A mutation. (A) Population r800 data plotted as a function of test-pulse potential reveals reduced inactivation at higher voltages. Each dot, mean ± SEM. n = 7 cells. (B) A two-pulse protocol is used to dissect voltage dependence of inactivation. A brief 15-ms prepulse to 10 mV quantifies available current prior to inactivation. Subsequently, a family of 800-ms voltage steps (test pulse) is used to evoke steady-state inactivation and a 15-ms postpulse is then used to quantify the current remaining following VDI. The extent of inactivation is quantified as the ratio of peak currents during postpulse to prepulse. Exemplar currents at three different test pulse potentials (light gray, −50 mV; gray, 0 mV; and black +50 mV) are shown. Exemplar trace for wild-type CaV1.3. Further analysis in Fig. 2, C and E. (C) Exemplar trace for DIV W[+2]A mutation. Further analysis in Fig. 2, D and F. (D–F) VDI of DIV W[+2]A probed in the presence of Ca2+ as charge carrier. Panel D, exemplar trace. Panel E, comparison of Ca2+ currents during prepulse (left, I1) versus postpulse (right, I2). Note that the current magnitude during the postpulse is…

Figure 3.. DIV W[+2]A mutation increases local conformational flexibility near the selectivity filter. (A) MD simulations were used to estimate the local conformational flexibility of the DIV pore helices of wild-type CaV1.3 embedded in a lipid bilayer. Colormap indicates computed B-factors for residues in DIV pore helices ranging from 0 (green) to 147.7 (red) corresponding to increasing flexibility. (B) DIV W[+2]A mutation increases local conformational flexibility. (C and D) In comparison to wild-type channels (panel C), simulations of DI W[+2]A mutant (panel D) show minimal change in computed B-factors for residues in DII pore helices. (E and F) Both wild-type (panel E) and DII W[+2]A mutant (panel F) show minimal change in local conformational flexibility. (G and H) Similarly, both wild-type (panel G) and DII W[+2]A mutant (panel H) exhibit minimal change in local conformational flexibility. This differential effect between DI–III versus DIV points to a specific role for DIV in tuning VDI. (I) Left: Comparison of wild-type versus DIV W[+2]A mutation reveals key changes in the selectivity filter conformation. The DIV W[+2]A mutation alters the arrangement of Ca2+ coordinating E[0]…

Figure 4.. Bulkiness of the DIV W[+2] residue tunes VDI. (A) Exemplar traces reveal intermediate inactivation of CaV1.3 DIV W[+2]T mutant (blue). Exemplar traces from wild-type (W[+2]) and DIV W[+2]A mutant channels are reproduced from Fig. 1 F to facilitate comparison. (B) VDI is similarly diminished for DIV W[+2]V mutation. Format as in panel A. (C) VDI is virtually absent for DIV W[+2]F mutation. (D) VDI is enhanced for DIV W[+2]G mutation. (E) Left: Structural model highlights residues in adjacent domains that are within 5 Å of W[+2] residue in a given domain. Right: Sequence alignment and residues in close vicinity of W[+2] residue. Neighborhood residues E[0], T[−2], and F[−6] are conserved across different domains. By contrast, DIV W[+2] is in the vicinity of DIII T[−5] while either a Q or an R is present at the −5 position of other domains. (F) Analysis of DIII T[−5]Q/DIV W[+2]A double mutation shows reduced VDI in comparison with DIV W[+2]A single mutation. (G) Population r800 values for DIV W[+2] mutations reveal a Boltzmann relation between VDI as the accessible side-chain surface area. DIII T[−5]Q/DIV W[+2]A reduced VDI in comparison to DIV W[+2]A mutation and deviates from this…

Figure 5.. Structural biparititioning of CDI mediated by distinct CaM lobes. (A) Exemplar currents show strong CDI of DI W[+2]A mutation as evident from accelerated current decay observed with Ca2+ (red) versus Ba2+ (black) currents evoked in response to a 300-ms test pulse. Ca2+ current decay of wild-type CaV1.3 is shown as rose trace to facilitate comparison. Here, measurement obtained in the presence of endogenous CaM and therefore net CDI includes contributions of both N- and C-lobes of CaM. (B and C) DII W[+2]A and DIII W[+2]A mutations minimally perturb CDI. Format as in panel A. (D) CaV1.3 DIV W[+2]A mutation partially diminishes CDI. Here, CDI is measured by comparing inactivation in Ca2+ with endogenous CaM versus CaM1234 coexpressed (blue trace). (E) Population data confirms a mild reduction in CDI observed with W[+2]A mutation. P < 0.001 by Tukey’s multiple comparisons test. Each bar and error, mean ± SEM. n = 7 cells for CaV1.3 wild-type and n = 5 for all mutant channels. (F–J) CDI mediated by the C-lobe of CaM is selectively diminished by CaV1.3 DIV W[+2]A mutation. Here, the component of C-lobe CDI is isolated by coexpression of CaM12. For DIV W[+2]A mutant, we compared…

Figure 6.. SF domain mutation enhances inactivation of TS-associated CaV1.2 mutant channel. (A) Top: Wild-type human CaV1.2 channel exhibits robust CDI and VDI. Black trace, Ba2+ current; red trace, Ca2+ current. Bottom: Population data show baseline inactivation of CaV1.2 measured as r300, the fraction of peak current remaining following 300 ms, with either Ca2+ (red) or Ba2+ (black) as the charge carrier. Each symbol, mean ± SEM. n = 5 cells. (B) CaV1.2 G406R mutant linked to TS shows reduced VDI and CDI. Format as in panel A. n = 6 cells. (C) Introducing the DIV W[+2]A mutation on TS-linked CaV1.2 G406R background enhances VDI. This finding suggests that increasing the conformational flexibility of DIV SF may be a potential approach to reverse TS pathophysiology. n = 6 cells.

Figure 7.. Asymmetric effect of SF mutations on CaV1.3 VDI and CDI. (A) Schematic shows asymmetric effect of the SF W[+2]A mutations in evoking VDI of CaV1.3. The interdomain interface between DIII and DIV is stabilized primarily by the conserved W[+2] residue which may serve as a wedge. Disruption of this interaction destabilizes the local conformation. By contrast, the interdomain interface between DI–DII, DII–DII, and DI–DIV is stabilized by both the W[+2] residue and through additional interdomain contacts. Consequently, SF conformation is maintained even with the W[+2]A mutation in DI through DIII. (B) For CDI, the W[+2] residue likely plays a role in transducing SF conformational rearrangements. The N- versus C-lobe of CaM appears to be asymmetrically coupled to distinct SF domains, likely evoking distinct SF conformational changes.