Mutations in the Kvβ2 binding site for NADPH and their effects on Kv1.4

Abstract

Kvβ2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kvβ2 binds NADP+, and it has been suggested that Kvβ2 is an oxidoreductase enzyme (1). To investigate how this function might relate to channel modulation, we made point mutations in Kvβ2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kvβ2 with Kvα1 channels. To characterize the Kvβ2 mutants functionally, we coinjected wild-type or mutant Kvβ2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kvβ2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kvβ2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kvβ2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kvβ2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kvβ2 may play a role in the processing of Kv1.4.