Asymmetric Contribution of a Selectivity Filter Gate in Triggering Inactivation of Ca V 1.3 Channels

Figures

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  Figure 1.. Asymmetric role of Domain IV pore helix in tuning VDI of Ca1.3. (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 (7UHG). 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-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 enhanced VDI…

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  Figure 2.. Distinct functional signature of CaV1.3 VDI. (A-B) Exemplar Ba2+ current traces evoked in response to a family of voltage-steps show minimal VDI of both wild-type CaV1.3 (panel A) and DIV W[+2]A mutant (panel B). CaM1234 co-expressed to exclude potential contribution of CDI. (C) A two-pulse protocol is used to quantify voltage-dependence of inactivation. Comparison of Ba2+ currents for wild-type CaV1.3 during the pre-pulse versus the post-pulse. CaV1.3 currents are increased in response to higher test-pulse potentials. See Fig. S2B for full exemplar trace. (D) DIV / W[+2]A mutant channels exhibit diminished peak currents during the post-pulse following test pulse to intermediate voltages. See Fig. S2C for full exemplar trace. (E) Analysis of wild-type CaV1.3 reveals minimal VDI across multiple voltages. Each dot and error, mean ± s.e.m. n = 7. (F) VDI of DIV / W[+2]A mutation exhibits a U-shaped voltage-dependence with strongest inactivation observed near 0 mV. Blue curve, voltage-dependence of inactivation obtained from whole cell normalized IV relations (Fig. S2E). Each dot and error, mean ± s.e.m. n = 7 cells. (G) Exemplar traces show similar kinetics of inactivation for DIV /…

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  Figure 3.. DIV W[+2]A mutation increases local conformational flexibility near the selectivity filter. (A) MD simulations were used to estimate local conformational flexibility of the domain IV 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-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-F) Both wild-type (panel E) and DII W[+2]A mutant (panel F) show minimal change in local conformational flexibility. (G-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 reveal key changes in the selectivity filter conformation. The DIV W[+2]A mutation alters the arrangement of Ca2+ coordinating E[0] residue, with DIV…

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  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 trances from wild-type (W[+2]) and DIV W[+2]A mutant channels are reproduced from Figure 1F 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 5Å 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 are 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 to 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…

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  Figure 5.. Structural biparitioning 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 – 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 co-expressed (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 ± s.e.m. n = 7 cells for CaV1.3 wild-type and n = 5 for all mutant channels. (F-J) CDI mediated by 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 co-expression of CaM12. For DIV W[+2]A mutant, we compare inactivation…

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  Figure 6.. SF domain mutation enhances inactivation of Timothy Syndrome associated CaV1.2 mutant channel. (A) Top, wild-ty pe 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 charge carrier. Each symbol, mean ± s.e.m. n = 5 cells. (B) CaV1.2 G406R mutant linked to Timothy syndrome 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.

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  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 are stabilized by both the W[+2] residue and through additional inter-domain 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 appear to be asymmetrically coupled to distinct SF domains, likely evoking distinct SF conformational changes.