Expression of tetraspanin protein CD63 enhances the PTK‐induced inhibition of ROMK channels

Abstract

Tetraspanin proteins have been shown to associate with activated PKC and to link PKC to its substates. In the present study, we examined the role of CD63 in mediating the effect of protein tyrosine kinase (PTK) on ROMK channels. Immunoprecipitation with CD63 antibody of lysates derived from renal outer medulla and cortex or HEK293 cells transfected with CD63 and c-Src led to co-precipitation of c-Src. We then used two electrode voltage clamp to study the effect of CD63 on ROMK channels in Xenopus oocytes. Although coexpression of CD63 had no effect on K currents in oocytes injected with ROMK1, it significantly enhanced the c-Src-induced inhibition of ROMK channels. The effect of CD63 on the cSrc-induced inhibition is not due to a decreased expression of ROMK1 channels, because inhibition of PTK with Herbimycin A abolished the inhibitory effect of c-Src on ROMK channels in oocytes injected with ROMK1+c-Src+CD63. Furthermore, coexpression of CD63 enhanced tyrosine phosphorylation of ROMK1 in HEK293 cells. Deletion of the last ten amino acids of the C-terminus of CD63, which contains an endocytosis motif, did not affect the association between c-Src and CD63. However, coexpression of the truncated CD63 abolished the inhibitory effect of c-Src on ROMK channels and decreased tyrosine phosphorylation of ROMK1 in comparison to that of HEK293 cells transfected with c-Src+ROMK1 or c-Src+ROMK1+CD63. Also, inhibition of PTK did not increase K currents in oocytes injected with ROMK1, c-Src and truncated CD63. We conclude that CD63 is associated with c-Src in the kidney and that coexpression of CD63 enhances the tyrosine phosphorylation of ROMK channels.