Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating

Yousra El Ghaleb,Monica L Fernández-Quintero,Stefania Monteleone,Petronel Tuluc,Marta Campiglio,Klaus R Liedl,Bernhard E Flucher

doi:10.1016/j.bpj.2021.09.004

Yousra El Ghaleb, Monica L Fernández-Quintero + Show 5 more

Open Access

https://doi.org/10.1016/j.bpj.2021.09.004

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Abstract

The voltage-gated calcium channel CaV1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of CaV1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire CaV1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for CaV1.1 and CaV1.2 L-type calcium channels. Moreover, in CaV1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-CaV1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-CaV1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V1/2, whereas no further shift of V1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in CaV1.1.

Highlights

Activation of voltage-gated cation channels is controlled by a set of four voltage-sensing domains (VSDs), symmetrically arranged around a common ion conduction pore (Fig. 1, A and B) [1]
In our ongoing efforts to understand the molecular mechanisms underlying the specific properties of the CaV1.1 VSDs and their differential contribution to regulating Ltype calcium currents and EC coupling, we recently identified an ion-pair partner of the outer gating charges (R1/R2) of VSD IV that is of critical importance for the regulation of the gating properties by alternative splicing [4]
The structure of the extracellular loop connecting S3 and S4 in the fourth VSD was modeled without the 19 amino acids encoded by the alternatively spliced exon 29, to generate the structure of the embryonic splice variant CaV1.1e

Summary

Introduction

Activation of voltage-gated cation channels is controlled by a set of four voltage-sensing domains (VSDs), symmetrically arranged around a common ion conduction pore (Fig. 1, A and B) [1]. Helix is moved outward relative to S1–S3, and the ensuing conformational change is transmitted to opening of the channel gate via a cytoplasmic S4-S5 linker. In this process, evenly spaced arginines and lysines in S4 (termed gating charges, R1, R2, .) are sequentially translocated across the membrane electric field, which is highly focused at the hydrophobic constriction site in the center of the VSD [5]. The energetically unfavorable movement of the positively charged amino acids through the hydrophobic environment is supported by the transient formation of ion pairs between the gating charges and negative countercharges in the S2 and S3 helices (called charge-transfer center or inner negative cluster). Additional ion pairs between the gating charges and countercharges in an extracellular negative cluster (ENC) help stabilize VSDs in successive resting and activated states and contribute to shaping the characteristic gating properties of different channels [6,7]

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