TYROSINE PHOSPHORYLATION MODULATES CURRENT AMPLITUDE AND KINETICS OF A NEURONAL VOLTAGE-GATED POTASSIUM CHANNEL

The modulation of the Kv1.3 potassium channel by tyrosine phosphorylat ion was studied. Kv1.3 was expressed in human embryonic kidney (HEK 29 3) cells, and its activity was measured by cell-attached patch recordi ng. The amplitude of the characteristic C-type inactivating Kv1.3 curr ent is reduced by >95%, in all cells tested, when the channel is co-ex pressed with the constitutively active nonreceptor tyrosine kinase, v- Src. This v-Src-induced suppression of current is accompanied by a rob ust tyrosine phosphorylation of the channel protein. No suppression of current or tyrosine phosphorylation of Kv1.3 protein is observed when the channel is co-expressed with R385A v-Src, a mutant with severely impaired tyrosine kinase activity. v-Src-induced suppression of Kv1.3 current is relieved by pretreatment of the HEK 293 cells with two stru cturally different tyrosine kinase inhibitors, herbimycin A and genist ein. Furthermore, Kv1.3 channel protein is processed properly and targ eted to the plasma membrane in v-Src cotransfected cells, as demonstra ted by confocal microscopy using an antibody directed against an extra cellular epitope on the channel. Thus v-Src-induced suppression of Kv1 .3 current is not mediated through decreased channel protein expressio n or interference with its targeting to the plasma membrane. v-Src co- expression also slows the C-type inactivation and speeds the deactivat ion of the residual Kv1.3 current. Mutational analysis demonstrates th at each of these modulatory changes, in current amplitude and kinetics , requires the phosphorylation of Ky 1.3 at multiple tyrosine residues . Furthermore, a different combination of tyrosine residues is involve d in each of the modulatory changes. These results emphasize the compl exity of signal integration at the level of a single ion channel.