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

Abstract

BK channels are dually regulated by voltage and Ca, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca 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 Ca 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 Ca. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca 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 Ca 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 Ca binding sites regulating the Ca 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.

Coupling of Ca2+and voltage activation in BK channels through the αB helix/voltage sensor interface

“…S1, and Table S2). Both the Ca 2+ bowl and RCK1 Ca 2+ site were found to contribute to Ca 2+ sensitivity in WT channels, as previously observed (7,48). The L390P mutation then reduced the contribution from both of these sites ( Fig.…”

“…In the absence of L390P mutations, our observations that RCK1 Ca 2+ sites acting alone make a greater contribution to Ca 2+ activation than Ca 2+ bowl sites acting alone, and that the sum of ΔV1/2 0→100 µM Ca for RCK1 Ca 2+ sites alone and Ca 2+ bowl sites alone can exceed ΔV1/2 0→100 µM Ca for WT channels has been described previously (47,48). Also observed by others have been equal contributions of the RCK1 Ca 2+ site and Ca 2+ bowl to left shifts in V1/2 (7,8,49), and equal contributions of these sites to decreasing the free energy necessary to activate voltage sensors (43).…”

“…Further support for possible common transduction elements comes from the structural observations that Ca 2+ bowl coordination is completed through contribution of aN438 (hN499) from the RCK1 N-lobe of an adjacent subunit, and that the RCK1 Ca 2+ site is completed through aD356 (hD367) borrowed from the Nlobe of the same subunit, such that the two high affinity sites may work jointly to tilt the N-lobe laterally and upward as a common transduction element for the RCK1 and Ca 2+ bowl sites (16,17). Functional studies further indicate that hR514 from RCK1 may coordinate with Ca 2+ bowl hE902 and hY904, leading to additional potential common elements in the Ca 2+ bowl and RCK1 Ca 2+ site activation pathways (48).…”

Hydration Mimicry by Membrane Ion Channels

“…Yet, new ion transport proteins, structures, functions, and mechanisms of both new and old transport proteins are discovered almost daily. This makes the field of ion channels and transporters one of the most active in molecular biology (15,35,41,68,69,70,71,72,73,74,75,76,77,78,79,80).…”

Physiological Roles and Therapeutic Potential of Ca2+ Activated Potassium Channels in the Nervous System

“…Previous considerations should be revisited in light of these novel findings, which undoubtedly set an unprecedented ground for SK channels drug discovery. These new IK structures appear barely 1 year after the cryo-EM structures of the full-length BK channel in the open and closed states was obtained ( Hite et al, 2017 ; Tao et al, 2017 ), revealing new and interesting insights in the channel structure–function relationships ( Giraldez and Rothberg, 2017 ; Zhou et al, 2017 ; Kshatri et al, 2018 ). In this context, other techniques such as patch-clamp fluorometry ( Zheng and Zagotta, 2003 ; Wulf and Pless, 2018 ) have proven to be useful tools to study conformational changes between subunits at the level of the gating ring during activation of functional BK channels ( Giraldez et al, 2005 ; Miranda et al, 2013 , 2016 ), and could also be extended to study other members of the K Ca subfamily.…”