Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4

Kv beta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kv beta 2 binds NADP(+), and it has been suggested that Kv beta 2 is an oxidoreductase enzyme (). To investigate how this function might relate to channel modulation, we made point mutations in Kv beta 2 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 beta 2 with Kv alpha 1 channels. To characterize the Kv beta 2 mutants functionally, we coinjected wild-type or mutant Kv beta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv beta 2 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 beta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta 2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kv beta 2 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 beta 2 may play a role in the processing of Kv1.4.