Atomic Force Microscopy Imaging Demonstrates that P2X2 Receptors Are Trimers but That P2X6 Receptor Subunits Do Not Oligomerize

Abstract

P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X 2 receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm 3 ) also points to a trimeric structure. P2X 2 receptors bearing His 6 epitope tags were incubated with anti-His 6 antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123°, again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X 6 receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm 3 . We conclude that P2X 2 receptors are trimers, whereas the P2X 6 receptor subunits do not form stable oligomers.P2X receptors contain an integral cation-selective ion channel that is opened by the binding of extracellular ATP (1, 2). Seven P2X receptor subunits have been identified, and these subunits associate together to form homo-or hetero-oligomeric receptors. Each subunit has two transmembrane regions (TMRs), 1 which are integrated into the membrane so that both N and C termini are intracellular. The large extracellular domain is glycosylated and contains several cysteine residues that form multiple disulfide bonds.The architecture of P2X receptors is still not clear, although the available evidence favors a trimeric arrangement of subunits, at least for some forms of the receptor. Early indications that the receptors were oligomers arose from electrophysiological studies of both endogenous receptors and exogenously expressed receptors, which revealed that receptor activation had a Hill coefficient greater than 1 (3, 4). Analysis of the mechanism of blockade of the T336C mutant P2X 2 receptor by methanethiosulfonate indicated a trimeric architecture (5). More recently, chemical cross-linking of P2X 1 , P2X 2 , and P2X 3 receptors again pointed to the existence of trimers (6, 7). It is fair to say, however, that this issue has not been completely resolved. For instance, under some circumstances, the P2X 1 , P2X 2 , and P2X 3 receptors have been found to migrate on non-denaturing gels as hexamers and even nonamers (6, 7). Further, the extracellular domain of the P2X 2 receptor behaves as a stable tetramer in solution (8). Finally, the P2X 6 receptor migrates as a tetramer on native gels (7).In the present study, we have combined a chemical crosslinking approach with direct imaging of P2X 2 and P2X 6 receptors by AFM. The ability to visualize single receptors afforded by AFM has permitted the measurement of the molecular volume of the receptors and the determination of the geometry of receptors liganded by antibodies against epitope tags present on the receptor subunits. Our data show convincingly that the P...

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