Fig. 2.

Key residues in TM1 and TM2 are required for authentic P2X2 trimeri sation. (A,B,D) HeLa cells were transiently transfected with plasmids expressing wild type rat P2X2 with a C-terminal GluGlu tag (wt) or a range of P2X2 point mutants. Alternatively, a plasmid encoding an HA-tagged 208 residue fragment of P2X2 lacking the second transmembrane domain was used (C). After harvesting of the cells, digitonin soluble material was analysed by blue native gel analysis in the absence (native) or presence of SDS (+SDS) as indicated. Following electrophoresis, samples were immunoblotted and analysed using antibodies recognising the GluGlu- (A,B,D) or HA-tag (C). The migration of native molecular weight markers (kDa) is indicated to the left of the immunoblots, and lanes sharing a common set of markers are derived from a single native gel. Individual lanes have been rearranged to facilitate direct comparisons. An asterisk (*) indicates the location of presumptive native P2X2 trimers, and an open circle the monomer resulting from SDS treatment. The open triangle indicates the migration of the P2X2-208 fragment after SDS treatment.