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Implications for Channel Gating
2 Istituto di Biofisica, Consiglio Nazionale delle Ricerche, I-16149 Genova, Italy
Address correspondence to Stefan Gründer, Department of Physiology II, Gmelinstr. 5, D-72076 Tübingen, Germany. Fax: 49-7071-29-5074; email: stefan.gruender{at}uni-tuebingen.de
Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 
3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.
Key Words: epithelial Na+ channel • ion channel • channel pore • Xenopus oocyte • channel gating
Abbreviations used in this paper: ASIC, acid-sensing ion channel; ENaC, epithelial sodium channel.
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