Binding of pyridine nucleotide coenzymes to the beta-subunit of the voltage-sensitive K+ channel.
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.
Liu
S Q
SQ
Division of Cardiology, University of Louisville, Jewish Hospital Heart and Lung Institute, Louisville, Kentucky 40202, USA.
Jin
H
H
Zacarias
A
A
Srivastava
S
S
Bhatnagar
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; C426239:Kcnab2 protein, rat; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D009249:NADP