Structural and Functional Analysis of the MthK K+ Channel RCK Domain

Abstract

Regulator of K+ conductance (RCK) domains control the activity of a variety of K+ channels and transporters, including the prokaryotic TrkA/H K+ transport complex and the eukaryotic BK channel, through binding of cytoplasmic ligands such as ATP, H+, and Ca2+. Thus RCK domains transduce ligand binding to gate transmembrane K+ flux in response to signaling events and cellular metabolism, in organisms ranging from bacteria to humans. In this work, I utilize the prokaryotic RCK domain containing K+ channel, MthK as a model system to provide insight toward the structural basis of ion channel gating by RCK domains. In MthK, binding of Ca2+ to an octameric ring of RCK domains (the gating ring) which is tethered to the pore of the channel, leads to a series of conformational changes that facilitates channel opening and K+ conduction. Using electrophysiology and X-ray crystallography, I identify the presence of additional Ca2+ binding sites in the MthK RCK domain, showing that each RCK domain contributes to three different regulatory Ca2+ binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca2+ binding sites, resulting in a stoichiometry of 24 Ca2+ ions per channel, is consistent with the steep relation between [Ca2+] and MthK channel activity. Comparison of Ca2+ bound and unliganded RCK domains suggests a physical mechanism for Ca2+-dependent conformational changes that underlie gating in this class of channels. To gain insight toward mechanisms of RCK domain activation, I crystallized and solved the structure of the RCK domain of MthK bound with Ba2+. The Ba2+-bound RCK domain was assembled as an octomeric gating ring, as observed in structures of the full-length MthK channel, and shows Ba2+ bound at several positions, one of which overlaps with a known Ca2+ binding site. Functionally, I determined that Ba2+ could activate reconstituted MthK channels as observed in electrophysiological recordings. These results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating. In an effort to more accurately describe the Ca2+-dependent gating process in MthK, I crystallized and solved structures of mutant and wild-type RCK domains, and found that distinct Ca2+ activation sites near the N- and C-termini of the RCK domain (termed C1 and C3, respectively) are allosterically coupled to one another, to affect tuning of Ca2+ affinity and Ca2+-dependent channel activation. These results define a structural mechanism of allosteric modulation in a ligand-gated K+ channel, and provide a framework for understanding similar mechanisms in related RCK-containing channels and transporters.