Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel

• Published in: Biochimica et biophysica acta. Biomembranes Vol. 1860; no. 4; pp. 943 - 952
• Main Authors: Kshatri, Aravind S., Gonzalez-Hernandez, Alberto J., Giraldez, Teresa
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.04.2018
• Subjects: BK channels; Ca2 + sensitivity; Electrophysiology; Free energy difference; Voltage dependence; Electrophysiology; Voltage dependence; Ca2+ sensitivity; BK channels; Free energy difference
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2017.09.023

BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as “gating ring”, which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. [Display omitted] •We have evaluated in human BK the role of residues involved in gating by Ca2+, recently proposed in the Aplysia structures.•Side chains of non-conserved residues G523 and E591, proposed to coordinate Ca2+ in Aplysia, have no functional role in hBK.•Functional data from hBK support an interaction between residues hR514 (RCK1 site), hE902 & hY904 (Ca2 + bowl site).•This interaction could provide a structural bridge underlying the cooperative mechanism of Ca2+ sensing in the BK channel.