Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Shizhen Wang,Yewande Alimi,Ailing Tong,Colin G Nichols,Decha Enkvetchakul

doi:10.1074/jbc.m807474200

Shizhen Wang, Yewande Alimi + Show 3 more

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https://doi.org/10.1074/jbc.m807474200

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Abstract

Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.

Highlights

Potassium channels are expressed in many cell types and are key players in a wide range of physiological processes
Purified KirBac1.1 Exists as a Tetramer in Detergent Solution—KcsA exists as a stable tetramer, even in the harsh conditions of SDS-PAGE [8, 17]
KirBac1.1 mutations with introduced cysteines, we found that substitution of cysteine at Ile-131 greatly enhanced the tetramer stability of KirBac1.1, as indicated by a major band at ϳ160 kDa, corresponding to the expected tetramer molecular mass, in SDS-PAGE (Fig. 1A)

Summary

Introduction

Potassium channels are expressed in many cell types and are key players in a wide range of physiological processes. The selectivity filter of KcsA, coordinated with Kϩ ions, can serve as a bridge between the four monomers to maintain the structure of the selectivity filter and the tetrameric architecture of the channel as a whole [11, 21]. Blocking ions, such as Ba2ϩ, act as strong stabilizers [17]. Because KcsA and KirBac1.1 are structurally similar in the transmembrane region of the pore, we hypothesized that permeant and blocking ions would affect KirBac1.1 tetramer stability in SDS-PAGE. The effects of blocking ions such as Ba2ϩ and Mg2ϩ on KirBac1.1 tetramer stability were examined to provide insight to the physical nature of their interaction with KirBac1.1, in the selec-

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