MEMBRANE TOPOLOGY OF AN ATP-GATED ION-CHANNEL (P2X RECEPTOR)

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X(2) receptor (about 65 kDa ) was reduced by pretreatment with endoglycosidase H. Mutagenesis of o ne or more of three potential asparagines (N182S, N239S, and N298S) fo llowed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by recepto rs. lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduce d into the W-terminal region (asparagine at position 9, 16, or 26) wer e not glycosylated, Asparagines were glycosylated when introduced at t he C-terminal end of the first hydrophobic domain (positions 62 and 66 ) and at the N-terminal end of the second hydrophobic domain (position 324), A protein in which the C terminus of one P2X(2) subunit was joi ned to the N terminus of a second P2X(2) subunit (from a concatenated cDNA) had twice the molecular mass of the P2X(2) receptor subunit, and formed fully functional channels. The experiments provide direct evid ence for the topology originally proposed for the P2X receptor, with i ntracellular N and C termini, two membrane-spanning domains, and a lar ge extracellular loop.