Subunit Oligomerization of Human ASIC1a in Xenopus Laevis Oocytes

Abstract

The crystal structure of the chicken Acid Sensing Ion Channel 1 (ASIC1) shows a homotrimeric channel complex. Because of conflicting results regarding the subunit stoichiometry of other members of the ASIC ion channel family, we have determined the mass of the functional human ASIC1a channel complex expressed in Xenopus oocytes. We have used sulfhydryl crosslinkers or oxidizing agents to stabilize the native oligomeric ASIC1a complex. In the cut-open oocyte recording system, the intracellular perfusion of 2-20 mM sodium tetrathionate (NaTT) did not affect hASIC1a activity. Western blot analysis shows that NaTT induces a shift in the molecular weight of ASIC1a from the monomeric 70 kDa, to higher molecular weights of 140 and 280 kDa. ASIC1a purification after surface biotinylation and crosslinking shows that the major form of ASIC1a at the plasma membrane corresponds to a 280 kDa channel complex. The use of different sulfhydryl crosslinkers allowed to stabilize on Western blot oligomeric ASIC1a complexes of 280, 210 and 140 kDa corresponding to tetramers, trimers and dimers. This subunit crosslinking of the ASIC1a complex depends on cysteines in the C-terminus of the protein. We confirmed by size exclusion chromatography the presence of a hASIC1a channel complex of a mass corresponding approximatively to 280 kDa. In Xenopus oocytes sulfhydryl crosslinkers or oxidizing reagents stabilized a hASIC1a complex of 280 kDa without affecting channel activity; the mass of this channel complex cannot simply account for a trimer. The C-terminus of ASIC1a subunits is involved in subunit-subunit interactions and may be important for the oligomerization of the functional channel complex. This work was supported by a grant from the SNF.