Tyrosine phosphorylation downregulates a potassium current in rat olfactory bulb neurons and a cloned Kv1.3 channel.

We are studying the regulation of ion channels by protein tyrosine kinases (TKs) using olfactory bulb neurons (OBNs) and a cloned voltage-dependent potassium channel, Kv1.3, as models. Rat OBNs, which express Kv1.3 channels, had whole-cell outward currents that were suppressed by picomolar quantities of margatoxin and showed a slow inactivation that increased over a 10-min period. These same pharmacological and kinetic properties were found also in human embryonic kidney (HEK 293) cells transfected with rat Kv1.3 cDNA. The insulin receptor TK was detected in the OB by Western analysis. Exogenous application of insulin was found to suppress whole-cell outward current in all OBNs tested. Perfusion of the nonreceptor Src kinase likewise suppressed outward current. Current was not suppressed with heat-inactivated Src or when adenosine triphosphate (ATP) was excluded from the pipette solution. The membrane permeant tyrosine phosphatase inhibitor, pervanadate, was found to suppress current in a subset of neurons, implying the presence of an endogenous tyrosine kinase in these neurons. Using site-directed mutagenesis of six tyrosine residues contained within good recognition motifs for tyrosine phosphorylation, we constructed conservative Y to F mutations in the Kv1.3 cDNA and expressed the Kv1.3 channels in HEK 293 cells. We found that Tyr449 is a target for both the pervanadate- and Src-induced suppression of Kv1.3 current; Tyr111, 112 and 113 are also important for modulation by pervanadate; and Tyr137 is a target for modulation by Src. In summary, tyrosine phosphorylation of Kv1.3 and related channels may be involved in the modulation of OBN excitability.