Divalent cation and chloride ion sites of chicken acid sensing ion channel 1a elucidated by x-ray crystallography

Abstract

Acid sensing ion channels (ASICs) are proton-gated ion channels that are members of the degenerin/epithelial sodium channel superfamily and are expressed throughout central and peripheral nervous systems. ASICs have been implicated in multiple physiological processes and are subject to numerous forms of endogenous and exogenous regulation that include modulation by Ca2+ and Cl- ions. However, the mapping of ion binding sites as well as a structure-based understanding of the mechanisms underlying ionic modulation of ASICs have remained elusive. Here we present ion binding sites of chicken ASIC1a in resting and desensitized states at high and low pH, respectively, determined by anomalous diffraction x-ray crystallography. The acidic pocket serves as a nexus for divalent cation binding at both low and high pH, while we observe divalent cation binding within the central vestibule on the resting channel at high pH only. Moreover, neutralization of residues positioned to coordinate divalent cations via individual and combined Glu to Gln substitutions reduced, but did not extinguish, modulation of proton-dependent gating by Ca2+. Additionally, we demonstrate that anion binding at the canonical thumb domain site is state-dependent and present a previously undetected anion site at the mouth of the extracellular fenestrations on the resting channel. Our results map anion and cation sites on ASICs across multiple functional states, informing possible mechanisms of modulation and providing a blueprint for the design of therapeutics targeting ASICs.

Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type

“…A Cl − binding site in close proximity of residue 212 was found in the open and desensitized but not in the closed ASIC1a structure (Jasti et al, 2007; Gonzales et al, 2009; Baconguis and Gouaux, 2012; Baconguis et al, 2014; Yoder and Gouaux, 2018; Yoder et al, 2018). It was shown that the chloride concentration affects the ASIC current decay kinetics (Kusama et al, 2010, 2013), and that mutations of the predicted Cl − binding site disrupted the modulation of the current decay kinetics if introduced in ASIC1a (Kusama et al, 2010), and had a partial, or no effect if introduced into ASIC2a or ASIC3, respectively (Kusama et al, 2013).…”

Conserved His-Gly motif of acid-sensing ion channels resides in a reentrant ‘loop’ implicated in gating and ion selectivity

“…In contrast, SMA-cASIC1a particles maintained at pH 8.0 occupy a high pH resting conformation, characterized by an expanded acidic pocket (Figure 1G) that resembles the high pH, resting state structures solved by x-ray crystallography and single particle cryo-EM (27). We propose that the limited resolution of the resting channel structure, while presumably impacted by sample conditions including thicker ice, may also be due to structural flexibility inherent to the resting channel conformation in the absence of divalent cations, which serve to stabilize an expanded acidic pocket at high pH (40) but which are incompatible with current SMA-based purification strategies.…”

Nociceptor Signalling through ion Channel Regulation via GPCRs

“…Only ASIC2b does not form functional homomeric channels [87]. One subunit consists of two transmembrane domains, having both the N- and C-termini at the intracellular side [88,89]. Most of the protein is located at the extracellular side forming the large extracellular domain (termed ECD).…”

“…ASIC channels follow a three-state kinetic model, from a closed to an activated to an inactivated state. The recovery from inactivation can only be achieved in high pH conditions [88] and this desensitized state is thought to be regulated by the thumb domain within the ECD [90].…”