Binding of pyridine coenzymes to the beta-subunit of the voltage sensitive potassium channels.
The beta-subunit of the voltage-sensitive K(+) channels shares 15-30% amino acid identity with the sequences of aldo-keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K(v)beta2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K(D) values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1+/-0.007 and 0.05+/-0.006 M, respectively - indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the alpha-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K(D) for NADH and NAD were between 1-3 microM whereas the K(D) for FAD was >10 microM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2' phosphate at the binding site. The selectivity of K(v)beta2.1 for NADPH over NADP may be significant in regulating the K(+) channels as a function of the cellular redox state.
Liu
S Q
SQ
Division of Cardiology, Department of Medicine, Jewish Cardiovascular Research Center, University of Louisville, 500, South Floyd Street, 40202, Louisville, KY, USA. siqiliu@louisville.edu
Jin
H
H
Zacarias
A
A
Srivastava
S
S
Bhatnagar
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D051668:Delayed Rectifier Potassium Channels; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits; D011725:Pyridines / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003067:Coenzymes; D017931:DNA Primers; D051668:Delayed Rectifier Potassium Channels; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits; D011725:Pyridines; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP