Tyrosine phosphorylation modulates current amplitude and kinetics of a neuronal voltage-gated potassium channel.
The modulation of the Kv1.3 potassium channel by tyrosine phosphorylation was studied. Kv1.3 was expressed in human embryonic kidney (HEK 293) cells, and its activity was measured by cell-attached patch recording. The amplitude of the characteristic C-type inactivating Kv1.3 current is reduced by >95%, in all cells tested, when the channel is co-expressed with the constitutively active nonreceptor tyrosine kinase, v-Src. This v-Src-induced suppression of current is accompanied by a robust 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 structurally different tyrosine kinase inhibitors, herbimycin A and genistein. Furthermore, Kv1.3 channel protein is processed properly and targeted to the plasma membrane in v-Src cotransfected cells, as demonstrated by confocal microscopy using an antibody directed against an extracellular epitope on the channel. Thus v-Src-induced suppression of Kv1.3 current is not mediated through decreased channel protein expression or interference with its targeting to the plasma membrane. v-Src co-expression also slows the C-type inactivation and speeds the deactivation of the residual Kv1.3 current. Mutational analysis demonstrates that each of these modulatory changes, in current amplitude and kinetics, requires the phosphorylation of Kv1.3 at multiple tyrosine residues. Furthermore, a different combination of tyrosine residues is involved in each of the modulatory changes. These results emphasize the complexity of signal integration at the level of a single ion channel.
Fadool
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DA
Department of Biochemistry and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254, USA.
Holmes
T C
TC
Berman
K
K
Dagan
D
D
Levitan
I B
IB
D018076:DNA, Complementary / Q000096:biosynthesis; D004594:Electrophysiology; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010766:Phosphorylation; D015221:Potassium Channels / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D018076:DNA, Complementary; D007202:Indicators and Reagents; D015221:Potassium Channels; D014443:Tyrosine; D015688:Oncogene Protein pp60(v-src)