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

Si-Qi Liu,Hongjun Jin,Albert Zacarias,Sanjay Srivastava,Aruni Bhatnagar

doi:10.1074/jbc.m008259200

Abstract

The beta-subunit of the voltage-sensitive K(+) (K(v)) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of K(v)beta2 shows NADP bound in its active site. Here we report that K(v)beta2 displays a high affinity for NADPH (K(d) = 0.1 micrometer) and NADP(+) (K(d) = 0.3 micrometer), as determined by fluorometric titrations of the recombinant protein. The K(v)beta2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, the K(d)(NADPH) of Q214R was 10-fold greater than the wild-type protein. The K(d)(NADPH) 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 K(d)(NADPH), shifted the pK(a) of K(d)(NADPH) 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 K(v)beta2-nucleotide complexes may be formed in vivo.

Highlights

The voltage-sensitive Kϩ (Kv) channels participate in several cellular processes
The results of this study show that the ␤-subunit of the Kv channel preferentially binds NADPH, suggesting that NADPH may be the most probable ligand bound to Kv␤ in vivo
Our data further show that NADPH binds to the C terminus or the conserved aldo-keto reductase (AKR) core of the protein and that this binding is not affected by the variable N terminus of the protein

Summary

EXPERIMENTAL PROCEDURES

Construction of the Expression Vector for Kv␤2—The cDNA containing the coding sequence for Kv␤2 was a gift from Dr Min Li. Data Analysis—Fluorescence titration data were fitted to a binding equation that takes into account the corrections for scatter, dilution, and cofactor absorbance [15]. In this equation, the fluorescence intensity I is a function of the cofactor concentration X, the protein concentration P, and the dissociation constant Kd, as shown below. The fluorescence intensity I is a function of the cofactor concentration X, the protein concentration P, and the dissociation constant Kd, as shown below In this relationship, Ymin and Ymax are the minimum and maximum fluorescence intensities, respectively, Ybgnd is the intensity of the background scatter, ␥ is Vinitial/( Vinitial ϩ VX) (the dilution factor), and ␳ is the absorbance coefficient of the cofactor. The best fit to the data was chosen on the basis of the standard error of the fitted parameter and the lowest value of ␴, which is the residual sum of squares divided by the degrees of freedom

RESULTS

NADPH NADPϩ

DISCUSSION