The Role of a Voltage-Dependent Ca²⁺Channel Intracellular Linker: A Structure-Function Analysis

Abstract

Voltage-dependent calcium channels (VDCCs) allow the passage of Ca 2ϩ ions through cellular membranes in response to membrane depolarization. The channel pore-forming subunit, ␣1, and a regulatory subunit (Ca V ␤) form a high affinity complex where Ca V ␤ binds to a ␣1 interacting domain in the intracellular linker between ␣1 membrane domains I and II (I-II linker). We determined crystal structures of Ca V ␤2 functional core in complex with the Ca V 1.2 and Ca V 2.2 I-II linkers to a resolution of 1.95 and 2.0 Å, respectively. Structural differences between the highly conserved linkers, important for coupling Ca V ␤ to the channel pore, guided mechanistic functional studies. Electrophysiological measurements point to the importance of differing linker structure in both Ca V 1 and 2 subtypes with mutations affecting both voltage-and calcium-dependent inactivation and voltage dependence of activation. These linker effects persist in the absence of Ca V ␤, pointing to the intrinsic role of the linker in VDCC function and suggesting that I-II linker structure can serve as a brake during inactivation.

CaV1.2 I-II linker structure and Timothy syndrome

“…This decrease is considerably larger than that found with G449R ( Fig. 5E in Almagor et al 1 ). Here an additive effect may be observable and the CDI of the double mutant, G436R/ Protein expression and purification.…”

“…2-7 TS mutant's effects occurred to a greater extent than in the other helix extending mutations, including both VDI and voltage dependence of activation (see Figure 5D, middle column in Almagor et al 1 ). Additionally, in line with our suggestion for a common role of the PL in VDI and CDI, our data present a similar trend with the substantially larger effect of G436R compared with G449R on CDI (Fig.…”

“…1 We observed distinct structural conformations for each of the channel subtype I-II linker/Ca V β complexes. Motivated by these structural Ca V 1.2 I-II linker structure and Timothy syndrome Lior This manuscript has been published online, prior to printing.…”

“…Helix-PL-AID proteins were expressed in E. coli and purified as described. 1 In a final size exclusion chromatography step t-test while comparison between several groups was done using a one-way ANOVA (Holm-Sidak) test, all with SigmaPlot (Systat Software).…”

Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1

“…Multiple molecular determinants for inactivation have been identified. However, unlike for some voltage-dependent K ϩ and Na ϩ channels (4), the dynamics of conformational changes that culminate in VDI and CDI are controversial and remain the subject of intense research (5)(6)(7)(8)(9)(10).…”