Double gaps alongShakerS4 demonstrate omega currents at three different closed states

Abstract

The aim of the present study was to investigate in detail how the voltage sensor in the Shaker potassium channel moves during the gating process. After the publication of the open channel structure from the crystallized K V Ap channel in 2003, an alternative so-called "paddle" model was put forward in contrast to the existing helical screw model. The voltage sensor s4 contains 4 arginine residues relevant for gating, R1(362), R2(365), R3(368) and R4(371), each separated by 2 neutral residues. These charged residues coil as one of three threads on the s4-α-helix. Based on a previous finding that the mutation R1s leads to the so-called omega leak current through a "gating-pore" in the closed state, we introduced gaps systematically along the arginine thread substituting long arginines by short serines. Mutations R2s or R3s did neither create transient nor steady leaks. The fact that the native residue A359, which is located three amino acids in front of R1, is a short one, motivated us to check its role. Mutation of A359 to arginine blocked the omega current in the R1s mutant indicating that the omega pore is occupied by A359 and R1. Introducing further double gaps (RR to ss) at sequential positions (0 + 1, 1 + 2, 2 + 3), produced clear leak currents which were remarkably stable over a wide voltage range. These leaks contradict that s4 would swing together with s3 in lipid according to the paddle hypothesis. Rather, our results show that during gating the s4 segment moves in 3 helical steps through a fixed pore formed by the channel protein.

The Pore of the Voltage-Gated Proton Channel

“…To permit an omega current through the Shaker K + channel VSDs requires a “double gap” (absence of arginine in two consecutive positions within S4’s repeating sequence of an arginine at every third position) [Gamal El-Din et al, 2010]. If this were also the case in Hv1, then substitution of N4 with arginine in the R3S background should prevent conduction of Gu + by bracketing the R3 position with arginines: the native R2 immediately before and an introduced R4 immediately after R3.…”

“…If R3 were positioned in the narrow part of the pore in the activated state, as its homologs are in the VSDs of Shaker [Larsson et al, 1996, Baker et al, 1998, Gamal El-Din et al, 2010] and Kv1.2 [Long et al, 2007] K + channels and Na + channels [Yang et al, 1996, Sokolov et al, 2005], it would be predicted to have a counter-charged partner from one of the other transmembrane segments. The partner residue for R3 in Kv1 channels—a conserved glutamate in the outer third of S2 (E283 in Shaker, E226 in Kv1.2) [Tiwari-Woodruff et al, 2000, Long et al, 2007]—is also present in Na + channels [DeCaen et al, 2008], but is missing in Hv1.…”

“…We searched for the Hv1 pore by seeking the portion of the VSD that confers ion selectivity. We began with a focus on S4 arginine positions because earlier work on the VSDs of K + and Na + channels showed that amino acid substitutions of one or more arginines creates an ion conducting pathway (also known as a “gating pore” or “omega pore”) through the VSD [Starace and Bezanilla, 2001, Starace and Bezanilla, 2004, Tombola et al, 2005, Sokolov et al, 2005, Sokolov et al, 2007, Tombola et al, 2007, Struyk et al, 2008, Gamal El-Din et al, 2010]. This suggested to us that a similar pathway could exist in the open state of the WT Hv1 channel to allow for proton permeation.…”

“…However, it appears that Shaker actually requires a double gap (substitution of two arginines) and that the outermost position (three residues before Shaker’s R1) is naturally “missing” (i.e. is an alanine), while an omega pore can also be made in Shaker at other voltages by mutations at neighboring pairs or arginines [Gamal El-Din et al, 2010]. A double gap is also needed to create an omega pore through the VSD in domain II of Na v 1.2a, [Sokolov et al, 2005].…”

Ion Channel Voltage Sensors: Structure, Function, and Pathophysiology

“…These results indicate that outward movement of the defective R2 and R3 gating charges into the gating pore upon activation causes an ionic leak through the voltage sensor, which is blocked by repolarization to return them to their resting position. Similarly, Gamal El-Din et al found that paired mutations of two arginine gating charges in Shaker K + channels to smaller residues are generally required to induce gating pore current (Gamal El-Din et al, 2009). The previously observed gating pore current measured for mutations of the R1 gating charge alone (Tombola et al, 2005) depends on the location of a small, uncharged amino acid residue (alanine) in the −3 position (R0) in the Shaker amino acid sequence (Gamal El-Din et al, 2009).…”