Proton Mediated Conformational Changes in ACID Sensing Ion Channel1a

Abstract

Acid sensing ion channels (ASIC) are cation channels, activated when there is a reduction in pH. They are involved in key functions including nociception, synaptic transmission and in the physiopathology of ischemic stroke. Previous crystal structures show a trimeric receptor, with each monomer having a large extracellular domain, shaped like a hand, with finger, palm and thumb domains which in turn connect to the transmembrane segments. Available structures indicate that there are three pairs of aspartate and glutamate residues located in the extracellular region, lining the thumb and finger domains. Previous studies have shown that mutating one or two pairs of the carboxylates leads to a shift in EC50 but not a loss in function, suggesting additional residues are involved in proton sensing. Our results from simulation studies indicate that neutralizing these two pairs alone is not sufficient to reduce the electrostatic potential. However, neutralizing all three pairs of carboxylates eliminates much of the negative electrostatic potential in this region, indicating the role of all three pairs in proton binding and gating of the ion channel. Electrophysiological studies with the triple mutant shows a loss of function, while surface biotinylation and pull down assays show expression of the receptors at the surface. To test the pH mediated structural changes and also the role of these residues in the same, we used Luminescence Resonance Energy Transfer to study the angstrom level changes that occur upon pH reduction from 8 to 6. We see that there is a cleft closure conformational change that occurs between the finger and thumb domain. This motion is lost in the triple carboxylate mutant protein, thus confirming that these residues play an important role in proton mediated conformational change in the receptor and ultimately gating of the receptor.