Tyrosine phosphorylation of the Kv1.3 potassium channel.
Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively 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 phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent 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 eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.
Holmes
T C
TC
Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254, USA.
Fadool
D A
DA
Levitan
I B
IB
D000818:Animals; D002417:Cattle; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D009154:Mutation; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics / Q000502:physiology*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D026902:Potassium Channel Blockers; D015221:Potassium Channels; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D010770:Phosphotransferases; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases