Subunit specificity of polyclonal antisera to the carboxy terminal regions of P2X receptors, P2X1 through P2X7

Polyclonal antisera generated to peptides corresponding to the carboxy termini of the seven cloned rat P2X receptors were compared using membranes from CHO-K1 or 1321N1 cells expressing recombinant rat or human P2X receptors (rat P2X(3), P2X(4), and P2X(5) and human P2X(1), P2X(2), P2X(6), and P2X(7)). Preimmune sera failed to recognize any bands in immunoblots against the membrane fractions. Antiserum to the P2X(1) receptor recognized a band of approximately 58 kDa while those to the rP2X(2) and the rP2X(3) receptors recognized doublets of approximately 60 and 64 kDa. The antiserum to the rP2X(4) receptor recognized a similarly sized doublet as well as a higher molecular weight species at the expected size for a receptor dimer. Antiserum to the rP2X(5) receptor revealed a single band at 64 kDa while antiserum to rP2X(6) and rP2X(7) gave single bands at 50 kDa and 95 kDa, respectively. In all cases, immunoreactivity was eliminated by preincubation of the antisera with the cognate peptide and there was no cross-reactivity of antisera with heterologous receptors. These antisera are useful reagents for the analysis of the P2X receptor subtypes in native tissues as well as for measuring receptor expression levels in transfected cell lines. (C) 1999 Wiley-Liss, Inc.