FIGURE 6.
Physical association of P2X4 and GABAA receptors and maintenance of current inhibition is dependent on specific residues of P2X4 C terminus in recombinant expression system. A, shown are representative Western blots with anti-FLAG or anti-myc antibodies after immunoprecipitation with anti-myc (IP: myc) or anti-FLAG antibodies (IP: FLAG) from extracts of cells expressing GABAA α2β3myc, P2X4FLAGIN, or P2X4FLAGINYV/ST alone or in combination (A1). Bars represent the relative intensities of the signals observed after immunoprecipitation by anti-myc or FLAG antibodies for both receptor types (Co-IP, co-immunoprecipitation fraction). Data are from four independent experiments; * p < 0.05 (A2). B, sequences of the C-terminal domain of wild-type P2X4 subunits and designed peptides (YV6 and YV6/ST). Adaptor protein 2 binding domain is in bold. C, shown is a Western blot with anti-myc antibody of oocytes expressing α2β3myc receptors after pull down by immobilized peptides (YV6 or YV6/ST) or in the absence of peptides (resin) (C1). Bars indicate that association of α2β3myc is significantly stronger with YV6 than with YV6/ST. * p < 0.05, three independent experiments (C2). ns, not significant. D, inward currents evoked with 100 μm ATP, GABA, or both agonists (Actual) in oocytes co-expressing P2X4FLAGIN and GABAA α2β3 receptors in the absence (Control) or after injection of YV6 peptide (150 μm) (D1). Current inhibition is reduced in the presence of YV6 peptide but not with YV6/ST. **, p < 0.005, n = 5–13. Vm = −60 mV (D2).