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

Abstract

Voltage-dependent Ca 2ϩ channels are membrane proteins that play a critical role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells (1-6). The Ca V ␣1 subunits of voltage-dependent Ca 2ϩ channels are evolutionarily related to the ␣ subunit of K V channels with a single polypeptidic chain carrying four domains of six transmembrane segments (S1-S6). Although the overall identity at the primary sequence level is quite low between Ca V and K V channels, it improves significantly for transmembrane segments. By homology with the three-dimensional structures of KcsA, MthK, K V AP, KirBac, and K V 1.2 channels (7-11), the four S4 transmembrane segments are thus believed to act as the voltage sensor, whereas the S6 helices line the channel pore of Ca V ␣1.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 (13-15).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 Domai...

Cooperative Activation of the T-type CaV3.2 Channel

“…⌬⌬G act,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 (39,51,52). The coupling or interaction energy ⌬⌬G inact,interact during channel inactivation was calculated as follows,…”

“…Functional Expression of Ca V 3.2-Oocytes were obtained from female Xenopus laevis clawed frogs as described previously (38,39,46). Oocytes were injected with 46 or 4.6 nl of a solution containing 1 g/l of cRNA coding for the Ca V 3.2 WT or mutant and incubated for 2 to 5 days after RNA injection.…”

“…Electrophysiological Recordings and Data AcquisitionWild-type and mutant channels were screened at room temperature for macroscopic Ba 2ϩ currents and for gating currents, with the cut-open oocyte voltage-clamp technique using a CA-1B amplifier (Dagan Corp., Minneapolis, MN) (47, 48) as described before (39). The membrane of the oocyte exposed to the bottom chamber was permeabilized by a brief treatment with 0.1% saponin.…”

“…Point mutations were produced with the QuikChange XLmutagenesis kit (Agilent Technologies, Santa Clara, CA) using 39-mer primers as described elsewhere (38,39,45). Constructs were verified by automated double-stranded sequence analysis (Genomics Platform, IRIC, Université de Montréal, QC, Canada).…”

“…In HVA Ca V 1.2, Ca V 2.1, and Ca V 2.3 channels, a cluster of hydrophobic residues in the distal S6 pore region were shown to stabilize the channel closed state (32)(33)(34)(35)(36)(37)(38)(39). In particular, we have shown that the conserved isoleucine residue in distal S6 was functionally coupled with a leucine residue at position 596 in the S4-S5 helix during the activation of HVA Ca V 2.3 (39). Whether each of the four domains moves independently or in a concerted fashion remains to be established.…”