Role of Cysteine Residues in the NCKX2 Na~+/Ca~(2+)-K~+ Exchanger:Generation of a Functional Cysteine-Free Exchanger

摘要

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 approx50% 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.