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A Residue at the Cytoplasmic Entrance of BK-Type Channels Regulating Single-Channel Opening by Its Hydrophobicity

Zhaohua Guo ^*, Caixia Lv ^*, Hong Yi , Yu Xiong ^*, Yingliang Wu , Wenxin Li , Tao Xu ^* and Jiuping Ding ^*

^* Key Laboratory of Molecular Biophysics (Huazhong University of Science and Technology), Ministry of Education, College of Life Science and Technology, Wuhan, Hubei 430074, China; and State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China

Correspondence: Address reprint requests to Jiuping Ding, Key Laboratory of Molecular Biophysics, Huazhong University of Science and Technology, Ministry of Education, College of Life Science and Technology, Wuhan, Hubei 430074, China. Tel.: 86-27-8779-2153; Fax: 86-27-8779-2024; E-mail: jpding{at}mail.hust.edu.cn; or to Tao Xu at the same address. Tel.: 86-10-6488-8469; Fax: 86-10-6486-7566; E-mail: xutao{at}ibp.ac.cn.

Single large-conductance calcium-activated K⁺ (BK) channels encoded by the mSlo gene usually have synchronous gating, but a Drosophila dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A) exhibits very flickery openings. To probe this difference in gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to those of dSlo1A. Rectification of the single-channel current-voltage relation of I323T decreased as [Ca²⁺ ]_in increased from 10 to 300 µM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating; i.e., increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic pore-lining residues at position 323 of mSlo act cooperatively in a "shutter-like" mechanism gating the permeation of K⁺ ions. Rate-equilibrium free energy relations analysis shows that the four I323 residues in an mSlo channel have a conformation 65% similar to the closed conformation during gating. Based on these observations, we suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.