Cleft Closure Mechanism in Gating of Acid Sensing Ion Channel 1A

Abstract

Acid sensing ion channels (ASICs) are trimeric proton activated channels which share sequence similarities with Degenerin/Epithelial sodium channels. They are responsible for proton evoked currents in central nervous system of most vertebrates. Their physiological functions include synaptic transmission, sensory perception, learning, and pathophysiology of stroke. Crystal structure of the low pH state of chicken ASIC shows thumb and palm like domains in the extracellular part of each subunit connected through hydrogen bonds between carboxylate residue pairs, thought to be part of proton sensors in ASIC. Based on the low pH structure it's been hypothesized that these domains would be further apart at high pH due to negative charges and protonation would allow the two domains to move closer, which in turn could trigger changes in the transmembrane segments, causing channel opening. We have used Fluorescence Resonance Energy Transfer(FRET) to study the high pH resting structure of ASIC and determined conformational changes between the thumb and palm domains. Cysteine residues were introduced on these domains and tagged with thiol reactive donor and acceptor fluorophores. The recognition site for protease Xa was introduced on either side of the cysteine on the palm domain. This allowed us to perform the FRET measurements in oocytes and HEK-293 cells without purification, as addition of the protease allowed us to quantitively characterize the background FRET. The FRET constructs were characterized using electrophysiology to establish that they were functional. The FRET based distances showed 3A decrease in distance between thumb domain residue 340 and palm domain residues 130 and 139 upon changing the pH from 8 to 6. This decrease in distance provides evidence for the hypothesized movement in the extracellular domain of ASIC and also supports the protonation of the carboxylate pairs across these domains.