Role of cysteine residues in the NCKX2 Na+/Ca(2+)-K+ Exchanger: generation of a functional cysteine-free exchanger.
Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na(+)/Ca(2+)-K(+) exchanger plays a critical role in Ca(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca(2+) that enters rod and cone cells via the cGMP-gated channels. NCKX1 and NCKX2 contain five highly conserved cysteine residues. The objectives of this study were threefold: (1) to examine the importance of cysteine residues in NCKX2 protein function; (2) to examine their role in the interaction between NCKX2 and the CNGA subunit of the cGMP-gated channel; and (3) to generate a functional cysteine-free NCKX2 protein. The latter will facilitate structural studies taking advantage of the unique chemistry of the thiol group following insertion of cysteine residues at specific positions in the cysteine-free background. We generated a cysteine-free NCKX2 mutant protein that showed normal protein synthesis and processing and approximately 50% wild-type cation transport function. Cysteine residues were also not critical for the formation of NCKX2 homo-oligmers or NCKX2 hetero-oligomers with the CNGA subunit of the cGMP-gated channel. Our results appear to rule out a critical importance of an intramolecular disulfide linkage in NCKX2 protein synthesis and folding as had been reported before.
Kinjo
Tashi G
TG
Department of Physiology & Biophysics, Faculty of Medicine, Cellular and Molecular Neurobiology Research Group, University of Calgary, 3330 Hospital Drive, N.W. Calgary, Alberta, T2N 4N1, Canada.
Szerencsei
Robert T
RT
Winkfein
Robert J
RJ
Schnetkamp
Paul P M
PP
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011233:Precipitin Tests; D017949:Retinal Cone Photoreceptor Cells / Q000502:physiology; D012694:Serine / Q000737:chemistry; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013050:Spectrometry, Fluorescence
D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D012694:Serine; D003545:Cysteine; D000409:Alanine