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Abstract

The pore-forming subunits of the voltage-sensitive K+ channel (Kv) associate with ancillary β-subunits that regulate inactivation and voltage-dependence of the channel. The β-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant Kvβ2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine nucleotides regulate the Kvβ-mediated inactivation of K+ channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvα1.5 cDNA led to the appearance of the Kv1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with Kvβ1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, Kvβ1.3 co-transfected with Kvα1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both Kvα1.5 and Kvβ1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD+ in the internal solution of the patch pipette abolished Kvβ-induced inactivation of Kv1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with Kv1.5 alone, indicating that in the absence of Kvβ, NAD+ does not affect the activity of Kvα. The inactivating currents recorded from cells expressing both Kvα1.5 and Kvβ1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD+ impart different conformational states to the Kvβ protein and that only the NADPH bound Kvβ imparts inactivation to non-inactivating K+ currents. Thus, differential binding of pyridine nucleotide coenzymes to Kvβ could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD+/NADPH ratio is sensitive to oxygen concentration, the differential changes in Kvβ-mediated inactivation of K currents by NAD+ and NADPH could represent an oxygen-sensing mechanism.