Abstract
The voltage-gated H(+) channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1-S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer's cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process.
Highlights
Voltage-gated ion channels sense the membrane potential and gate ion permeation to generate electronic signals in many cell types [1]
Unique Trp at the Middle of the S4 Helix—Hv is a voltage sensor domain-like protein that was cloned on the basis of a homology search for the voltage sensor sequence of other voltage-gated channels [3, 4]
If the effect of Trp introduction on the gating kinetics is independent of the effect of channel dimerization, the ⌬⌬G changes caused by the two mutations will be additive
Summary
Voltage-gated Hϩ channel; NA-mutant, nonaromatic mutant; ANOVA, analysis of variance. Cooperative Deactivation by Trp in S4 of the Hv Dimer well tolerated at positions that are involved in tight proteinprotein interactions [25, 26], which raises the question of why a Trp is situated in a region of close S4-S4 interaction within the dimeric channel. The adjacency of the parallel S4 dipoles would seem to be energetically incompatible within a protein structure. Until now, these questions have not attracted much attention. To understand the functional and structural significance of the uniquely conserved Trp, we performed the electrophysiological analysis combined with the molecular dynamics simulation of WT and Trp mutants within Hv monomers and dimers. We show that the Trp residues form a direct -stacking within a dimer only when the channel is closing and produce the characteristic slow deactivation phase observed in the native Hv
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