In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct. Immunoprecipitation of tissue lysates from renal cortex and outer medulla or 293T cells transfected with CD63 reveals that CD63 was associated with RPTPalpha both in situ and in transfected cells. Expression of CD63 in 293T cells stimulated the phosphorylation of tyrosine residue 416 of c-Src but decreased the phosphorylation of tyrosine residue 527, indicating that expression of CD63 stimulates the activity of c-Src. Furthermore, c-Src was coimmunoprecipitated with RPTPalpha and CD63 both in 293T cells transfected with CD63 and in lysates prepared from native rat kidney. Potassium restriction had no effect on the expression of RPTPalpha, but it increased the association between c-Src and RPTPalpha in the renal cortex and outer medulla. We also used two-electrode voltage clamp to study the effect of CD63 on ROMK channels in Xenopus oocytes. Expression of CD63 had no significant effect on potassium currents in oocytes injected with ROMK1; however, it significantly enhanced the c-Src-induced inhibition of ROMK channels in oocytes injected with ROMK1+c-Src. The effect of CD63 on the c-Src-induced inhibition was not due to a decreased expression of ROMK1 channels, because blocking 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. We conclude that CD63 plays a role in the regulation of ROMK channels through its association with RPTPalpha, which in turn interacts with and activates Src family PTK, thus reducing ROMK activity.     «

In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct. Immunoprecipitation of tissue lysates from renal cortex and outer medulla or 293T cells transfected with CD63 reveals that CD63 was associated with RPTPalpha both in situ and in transfected cells. Express...     »