Abstract
Protonation of several amino acid residues in the extracellular domain (ECD) of acid-sensing ion channel (ASIC) causes conformational changes that lead to opening of the channel. It is not clear how conformational changes in ECD are coupled to channel gating. Here, we show that the loop connecting β9 and α4 at the base of the thumb region of ECD interacts with post-TM1 to stabilize the channel in the closed state. Flexibility of these two regions is important for optimum gating of the channel. In ASIC1a, when Y71 (post-TM1) and W287 (β9–α4 loop) were mutated to cysteine, they formed disulfide bond in the closed state. Breaking of the disulfide bond by reducing agent dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) potentiated the current significantly. Engineered cysteine G288C reacted with sulfhydryl-specific methanethiosulfonate ethyltrimethylammonium (MTSET) in the open state but not in closed/steady desensitized state, suggesting gating-associated conformational movement of this loop. We also identified a salt bridge between highly conserved R64 at TM1 and D432 at TM2 that is important for optimum gating.
Based on our results and other published work, we propose that proton binding in ECD is followed by the displacement of the β9–α4 loop of the thumb, leading to the rotation of TM1. Conformational movement propagates to TM2 and the channel gate opens by the concomitant movement of TM2 and breaking of the salt bridge between R64 and D432.
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Acknowledgments
This work was supported by the Department of Science and Technology, India. Sandip M. Swain received a doctoral fellowships from the University Grant Commission, India. We acknowledge Mrityunjay for helping with the computational graphics. Special thanks to Divya for her efforts in correcting the manuscript. We are grateful to Francois Rugiero (University College London, UK) for providing rASIC1a construct.
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Swain, S.M., Bera, A.K. Coupling of Proton Binding in Extracellular Domain to Channel Gating in Acid-Sensing Ion Channel. J Mol Neurosci 51, 199–207 (2013). https://doi.org/10.1007/s12031-013-9991-x
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DOI: https://doi.org/10.1007/s12031-013-9991-x