Molecular determinants of Ca2+ sensitivity at the intersubunit interface of the BK channel gating ring

Qin Li,Yunkun Wu,Jiusheng Yan,Yingxin Li,Alexandre G Vouga,Hao-Min Pan,Hua Wei,Brad S Rothberg

doi:10.1038/s41598-017-19029-8

Abstract

The large-conductance calcium-activated K+ (BK) channel contains two intracellular tandem Ca2+-sensing RCK domains (RCK1 and RCK2), which tetramerize into a Ca2+ gating ring that regulates channel opening by conformational expansion in response to Ca2+ binding. Interestingly, the gating ring’s intersubunit assembly interface harbors the RCK2 Ca2+-binding site, known as the Ca2+ bowl. The gating ring’s assembly interface is made in part by intersubunit coordination of a Ca2+ ion between the Ca2+ bowl and an RCK1 Asn residue, N449, and by apparent intersubunit electrostatic interactions between E955 in RCK2 and R786 and R790 in the RCK2 of the adjacent subunit. To understand the role of the intersubunit assembly interface in Ca2+ gating, we performed mutational analyses of these putative interacting residues in human BK channels. We found that N449, despite its role in Ca2+ coordination, does not set the channel’s Ca2+ sensitivity, whereas E955 is a determinant of Ca2+ sensitivity, likely through intersubunit electrostatic interactions. Our findings provide evidence that the intersubunit assembly interface contains molecular determinants of Ca2+-sensitivity in BK channels.

Highlights

X-ray crystallography of BKα C-termini in humans and zebrafish[20,21] and cryo-electron microscopy of the entire BKα channel in Aplysia californica[22,23] at near-atomic resolutions have revealed that the tandem RCK1 and RCK2 domains from four individual subunits are organized into a gating ring of eight RCK domains by tetramerization at the intersubunit assembly interface
To probe the role of the intersubunit assembly interface in BK channel Ca2+ gating, we perturbed the structure of the interface by eliminating intersubunit Ca2+ coordination mediated by N449 residues, and by disrupting intersubunit electrostatic interactions mediated by E955 residues, in human BK channels
E955 plays an important role in determining BK channel Ca2+-sensing likely through intersubunit electrostatic interactions, by a mechanism that is mediated in part through the Ca2+ bowl

Summary

Introduction

Our findings provide evidence that the intersubunit assembly interface contains molecular determinants of Ca2+-sensitivity in BK channels. Comparisons of the BK channel structures in the absence and presence of Ca2+ indicate that the gating ring expands in response to Ca2+ binding and propagates conformational changes to the channel pore[20,22], suggesting a likely involvement of the interfaces between the RCK domains of adjacent subunits, known as the “intersubunit assembly interface”[20,21], in BK channel activation by Ca2+. E955 plays an important role in determining BK channel Ca2+-sensing likely through intersubunit electrostatic interactions, by a mechanism that is mediated in part through the Ca2+ bowl

Methods

Results

Conclusion