KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain

Frank S Choveau,Nicolas Rodriguez,Fayal Abderemane Ali,Alain J Labro,Thierry Rose,Shehrazade Dahimène,Hélène Boudin,Carole Le Hénaff,Denis Escande,Dirk J Snyders,Flavien Charpentier,Jean Mérot,Isabelle Baró,Gildas Loussouarn

doi:10.1074/jbc.m110.146324

Abstract

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

Highlights

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1–S6) proteins
Our results show that S4-S5 linker (S4S5L) acts as an inhibiting ligand that binds with low affinity to S6 C terminus (S6T) only at negative potentials, locking the S5-S6 pore domain in a closed state
The state-dependent interaction between S4S5L and S6T has already been proposed by Ferrer et al [9] for hERG, another delayed rectifier potassium channel. This idea was suggested by the fact that a covalent link between S4S5L and S6T stabilizes the hERG closed state [9]

Summary

The abbreviations used are

S4S5L, S4-S5 linker; S6T, S6 C terminus; hERG, human ether-a-go-go-related gene; HCN, hyperpolarization-activated cyclic nucleotide-gated channel. The state-dependent interaction between S4S5L and S6T suggests that this interaction may stabilize the open state of hyperpolarization-activated channels (KAT1, HCN) and the closed state of depolarization-activated channels (hERG). Scanning mutagenesis studies were performed in a cardiac voltage-gated potassium channel, KCNQ1 [10, 11] to study the role of S4S5L (see companion article [31]) and S6T [12] in the voltage modulation of this channel. These studies identified residues implicated in the S4S5L/S6T interaction. Upon depolarization of the plasma membrane, S4 pulls S4S5L away from S6T, allowing channel opening

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION