Abstract
The acid-sensing ion channels (ASICs) open in response to extracellular acidic pH, and individual subunits display differential sensitivity to protons and calcium. ASIC1a acts as a high affinity proton sensor, whereas ASIC2a requires substantially greater proton concentrations to activate. Using chimeras composed of ASIC1a and ASIC2a, we determined that two regions of the extracellular domain (residues 87-197 and 323-431) specify the high affinity proton response of ASIC1a. These two regions appear to undergo intersubunit interactions within the multimeric channel to specify proton sensitivity. Single amino acid mutations revealed that amino acids around Asp(357) play a prominent role in determining the pH dose response of ASIC1a. Within the same region, mutation F352L abolished PcTx1 modulation of ASIC1a. Surprisingly, we determined that another area of the extracellular domain was required for calcium-dependent regulation of ASIC1a activation, and this region functioned independently of high affinity proton sensing. These results indicate that specific regions play overlapping roles in pH-dependent gating and PcTx1-dependent modulation of ASIC1a activity, whereas a distinct region determines the calcium dependence of ASIC1a activation.
Highlights
IBN-0416920. 1 Supported by American Heart Fellowship 0715513B. 2 To whom correspondence should be addressed: Dept. of Neuroscience, Mammals have four genes encoding acid-sensing ion channels (ASICs) that encode at least six different ASIC subunits [1,2,3, 10]
Two Regions of the Extracellular Domain Specify the pH Sensitivity of ASIC1a—ASIC1a is activated by pH 6.7 and shows a pH of half-maximal activation of ϳ6.5
Calcium Dependence Is Determined by Amino Acids 197– 323—The pH dose response of ASIC1a is markedly affected by extracellular calcium, with increasing concentrations of calcium increasing the concentration of protons required for channel activation [4, 27,28,29]
Summary
Construction of Chimeras, Site-directed Mutagenesis, and Expression in Xenopus Oocytes—Human ASIC1a- and ASIC2aencoding cDNAs in the pMT3 mammalian expression vector have been described previously [19, 20]. These sequences correspond to GenBankTM accession number NM_001095 for human ASIC1a and NM_001094 for human ASIC2a. The pH0.5 was calculated by fitting the data from individual pH dose-response experiments within each oocyte using the equation, 10n(pH Ϫ ), pH0.5). The variability in the calculated Hill coefficient of ASIC1a limited significance substantially. The number of the first amino acid within the break point motif of human ASIC1a is indicated (see “Experimental Procedures”).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
