Role of Cysteine Residues in the NCKX2 Na super(+)/Ca super(2+)-K super(+) 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 super(+)/Ca super(2+)-K super(+) exchanger plays a critical role in Ca super(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by...

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Published in:Biochemistry (Easton) 2004-06, Vol.43 (24), p.7940-7947
Main Authors: Kinjo, T G, Szerencsei, R T, Winkfein, R J, Schnetkamp, PPM
Format: Article
Language:English
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Summary:Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na super(+)/Ca super(2+)-K super(+) exchanger plays a critical role in Ca super(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca super(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 similar to 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.
ISSN:0006-2960
DOI:10.1021/bi049538y