Assembly of Retinal Rod or Cone Na super(+)/Ca super(2+)-K super(+) Exchanger Oligomers with cGMP-Gated Channel Subunits as Probed with Heterologously Expressed cDNAs

• Published in: Biochemistry (Easton) 2003-04, Vol.42 (15), p.4593-4600
• Main Authors: Kang, KyeongJin, Bauer, P J, Kinjo, Tashi G, Szerencsei, R T, Boenigk, W, Winkfein, R J, Schnetkamp, PPM
• Format: Article
• Language: English
• ISSN: 0006-2960
• DOI: 10.1021/bi027276z

Proper control of intracellular free Ca super(2+) is thought to involve subsets of proteins that co-localize to mediate coordinated Ca super(2+) entry and Ca super(2+) extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca super(2+) influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na super(+)/Ca super(2+)-K super(+) exchanger (NCKX) is the only Ca super(2+) extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments.