Binding of Pyridine Nucleotide Coenzymes to the β-Subunit of the Voltage-sensitive K+ Channel

Abstract

The β-subunit of the voltage-sensitive K+ (Kv) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of Kvβ2 shows NADP bound in its active site. Here we report that Kvβ2 displays a high affinity for NADPH (K d = 0.1 μM) and NADP+ (Kd = 0.3 μM), as determined by fluorometric titrations of the recombinant protein. The K vβ2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, the K dNADPH of Q214R was 10-fold greater than the wild-type protein. The KdNADPH was unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease in KdNADPH, shifted the pKa of K dNADPH from 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of Kvβ2-nucleotide complexes may be formed in vivo.