Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Li Yi,Stephen W Ragsdale,Jeffrey T Morgan

doi:10.1074/jbc.m110.116483

Li Yi, Stephen W Ragsdale + Show 1 more

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https://doi.org/10.1074/jbc.m110.116483

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Abstract

Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca(2+) and voltage-activated K(+) channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca(2+)-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K(d) = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K(d) = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.

Highlights

IntroductionGrant R21HL089837. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental “Methods,” equations, and Figs
Grant R21HL089837. □S The on-line version of this article contains supplemental “Methods,” equations, and Figs
Because our primary interests were in the effects of heme and redox on the channel and because the RCK domains can be found in other non-heme-regulated potassium channels, we focused on expressing the heme binding domain (HBD), which contains the characteristic CXXCH motif and connects RCK1 and RCK2 in the human BK channel

Summary

Introduction

Grant R21HL089837. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental “Methods,” equations, and Figs. One of the aims of this work is to further explore the interactions between the BK channel and HO2, heme, and CO This is significant because both heme and CO serve as signaling molecules in various physiological processes in cells, including circadian rhythm, oxygen utilization, and T cell activation [18, 19]. A segment that links RCK1 and RCK2 has been implicated in heme binding [14, 16] This linker region contains a CXXCH (X is any amino acid) motif that is highly conserved in all BK channels, and replacement of the Cys or His residues by Ser or Arg, respectively, abolish the sensitivity of the BK channel to heme and CO [14]. CO binds to the complex between heme and a synthetic 23-residue heme-binding peptide (HBP) containing residues 601– 623 of the BK channel and including

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