Requirement of Multiple Protein Domains and Residues for GatingKATP Channels by Intracellular pH

Hailan Piao,Ningren Cui,Haoxing Xu,Jinzhe Mao,Asheebo Rojas,Runping Wang,Latifat Abdulkadir,Lilly Li,Jiangping Wu,Chun Jiang

doi:10.1074/jbc.m106123200

Abstract

ATP-sensitive K(+) channels (K(ATP)) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K(ATP) by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating K(ATP) by protons are demonstrated. The pH sensitivity enables the K(ATP) to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.

Highlights

ATP-sensitive Kϩ channel (KATP)1 is a unique member in the Kϩ channel family, which directly couples the intermediary metabolism to cellular excitability [1, 2]
The pH sensitivity is independent of the SUR subunit and other cytosolic factors, suggesting that the pH sensing mechanisms are located in the Kir subunit [21]
We have demonstrated several protein domains and amino acid residues in the Kir6.2 necessary for the pH sensitivity of the KATP channels

Summary

Introduction

ATP-sensitive Kϩ channel (KATP) is a unique member in the Kϩ channel family, which directly couples the intermediary metabolism to cellular excitability [1, 2]. Our recent studies have shown that the cloned KATP responds to acidic pH [21] These channels are strongly stimulated by hypercapnia and intracellular acidosis. If the pH sensitivity is an inherent property of Kir proteins, there should be special structures responsible for channel gating by protons These structures are likely to be located in the Kir subunit, since the SUR subunit is not required for the pH sensitivity. Several chimerical channels were generated based on peptide sequences of Kir6.2 and Kir1.1, a Kir channel that is inhibited by intracellular acidosis [23,24,25] and has been shown to express functional channels in its chimeras with Kir6.2 [26] These chimeras were studied in whole-cell recording using hypercapnia, a condition that does not cause channel rundown [21]. Replacement of any of them with their counterpart residues in Kir1.1 interrupts the channel sensitivity to CO2/pH

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Conclusion