Assembly of Retinal Rod or Cone Na+/Ca2+-K+ Exchanger Oligomers with cGMP-Gated Channel Subunits as Probed with Heterologously Expressed cDNAs

详细信息    查看全文

• 作者：KyeongJin Kang ; Paul J. Bauer ; Tashi G. Kinjo ; Robert T. Szerencsei ; Wolfgang Bö ; nigk ; Robert J. Winkfein ; and Paul P. M. Schnetkamp
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：April 22, 2003
• 年：2003
• 卷：42
• 期：15
• 页码：4593 - 4600
• 全文大小：153K
• 年卷期：v.42,no.15(April 22, 2003)
• ISSN：1520-4995

文摘

Proper control of intracellular free Ca²⁺ is thought to involve subsets of proteins that co-localize to mediate coordinated Ca²⁺ entry and Ca²⁺ extrusion. The outer segments of vertebrate rod andcone photoreceptors present one example: Ca²⁺ influx is exclusively mediated via cGMP-gated channels(CNG), whereas the Na⁺/Ca²⁺-K⁺ exchanger (NCKX) is the only Ca²⁺ 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 rodouter segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation toexamine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either therod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNGinteractions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rodNCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predictedfor a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particularcysteine 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 couldinteract with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of thetwo large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggestingthat it is mediated by the highly conserved transmembrane spanning segments.