TYROSINE PHOSPHORYLATION OF THE KV1.3 POTASSIUM CHANNEL

Kv1.3, a voltage-dependent potassium channel cloned from mammalian bra in and T lymphocytes, contains multiple tyrosine residues that are put ative targets for tyrosine kinases. We have examined the tyrosine phos phorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Ty rosine phosphorylation is measured by a strategy of immunoprecipitatio n followed by Western blot analysis, using antibodies that specificall y recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutiv ely active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline ph osphatase before Western blot analysis, Coexpression with a receptor t yrosine kinase, the human epidermal growth factor receptor, also cause s an increase in tyrosine phosphorylation of Kv1.3, The effects of end ogenous tyrosine kinases were examined by treating Kv1.3-transfected c ells with the specific membrane-permeant tyrosine phosphatase inhibito r pervanadate. Pervanadate treatment causes a time- and concentration- dependent increase in the tyrosine phosphorylation of Kv1.3. This incr eased tyrosine phosphorylation of Kv1.3 is accompanied by a time-depen dent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminat ed by mutation of a specific tyrosine residue, at position 449 of Kv1. 3, to phenylalanine. Thus, there is a continual phosphorylation and de phosphorylation of Kv1.3 by endogenous kinases and phosphatases, and p erturbation of this constitutive phosphorylation/dephosphorylation cyc le can profoundly influence channel activity.