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

Kv beta2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kv beta2 binds NADP(+), and it has been suggested that Kv beta2 is an oxidoreductase enzyme (1). To investigate how this function might relate to channel modulation, we made point mutations in Kv beta2 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 Kv beta2 with Kv alpha1 channels. To characterize the Kv beta2 mutants functionally, we coinjected wild-type or mutant Kv beta2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv beta2 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 Kv beta2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta2 subunits with Kv1.4, Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing ef. feet of Kv beta2 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 Kv beta2 may play a role in the processing of Kv1.4.