Interaction Sites between the Slo1 Pore and the NH2 Terminus of the β2 Subunit, Probed with a Three-residue Sensor

Hui Li,Jing Yao,Xiaotian Tong,Zhaohua Guo,Ying Wu,Liang Sun,Na Pan,Houming Wu,Tao Xu,Jiuping Ding

doi:10.1074/jbc.m607063200

Abstract

Calcium- and voltage-gated (BK) K(+) channels encoded by Slo1 play an essential role in nervous systems. Although it shares many common features with voltage-dependent K(V) channels, the BK channel exhibits differences in gating and inactivation. Using a mutant in which FWI replaces three residues (FIW) in the NH(2) terminus of wild-type beta2-subunits, in conjunction with alanine-scanning mutagenesis of the Slo1 S6 segment, we identify that the NH(2) terminus of beta2-subunits interacts with the residues near the cytosolic superficial mouth of BK channels during inactivation. The cytosolic blockers did not share the sites with NH(2) terminus of beta2-subunits. A novel blocking-inactivating scheme was proposed to account for the observed non-competition inactivation. Our results also suggest that the residue Ile-323 plays a dual role in interacting with the NH(2) terminus of beta2-subunits and modulating the gating of BK channels.

Highlights

Ca2ϩ and voltage-gated Kϩ channels (BK channels)4 are encoded by mammalian Slo1 genes related to the Drosophila Slowpoke (Slo) gene [1, 2]
Evidence supporting the idea that the inactivation domain (ID) must insert into the pore is derived from the blocking experiments of KV channels by cytosolic blockers, which compete with the ID for channel occupancy [13, 14], resulting in the slowing of inactivation kinetics
With a mutant FWI, which is obtained from substituting the initial three amino acids (FIW) of the h␤2-subunit NH2 terminus with FWI, and a systematic alanine-scanning mutagenesis of the mSlo1 S6 segment, we demonstrate that the residue Ile323 of mSlo1 ␣-subunits plays a dual role in interacting with the ID of h␤2-subunits and modulating the gating of BK channels

Summary

Introduction

Ca2ϩ and voltage-gated Kϩ channels (BK channels)4 are encoded by mammalian Slo1 genes related to the Drosophila Slowpoke (Slo) gene [1, 2]. On the basis of our preliminary results, it is difficult to use the FIW to determine the interacting sites due to its doubtful time constants in recovery from inactivation (data not shown).

Results

Conclusion