title: Stoichiometry of the retinal cone Na/Ca-K exchanger heterologously expressed in insect cells: comparison with the bovine heart Na/Ca exchanger.
abstract: The transport stoichiometry is an essential property of antiporter and symporter transport proteins. In this study, we determined the transport stoichiometry of the retinal cone potassium-dependent Na/Ca exchanger (NCKX) expressed in sodium-loaded cultured insect cells. The Na/Ca and Rb/Ca coupling ratios were obtained by direct measurements of the levels of (86)Rb and (45)Ca uptake and sodium release associated with reverse Na/Ca exchange. Rb/Ca coupling ratios of 0.98 [standard deviation (SD) of 0.12, 15 observations] and 0.92 (SD of 0.12, 13 observations) were obtained for the chicken and human retinal cone NCKX, respectively. Na/Ca coupling ratios of 4.11 (SD of 0.24, 10 observations) and 3.98 (SD of 0.34, 15 observations) were obtained for the chicken and human retinal cone NCKX, respectively, whereas a lower average coupling ratio of 3.11 (SD of 0.34, 10 observations) was obtained with cells expressing the bovine Na/Ca exchanger (NCX1). These results are consistent with a 4Na/1Ca + 1K stoichiometry for retinal cone NCKX. High Five cells expressing full-length dolphin rod NCKX, Caenorhabditis elegans NCKX, or bovine rod NCKX from which the two large hydrophilic loops were removed all showed a significant calcium-dependent (86)Rb uptake, whereas no calcium-dependent (86)Rb uptake was observed in cells expressing bovine NCX1. The calcium dependence of (45)Ca uptake yielded values between 1 and 2.5 microM for the external calcium dissociation constant of the different NCKX proteins studied here.
authors.affiliation: Department of Physiology and Biophysics and The CIHR Ion Channel and Transporter Group, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, Northwest Calgary, Alberta T2N 4N1, Canada.
authors.forename: R T
authors.initials: RT
authors.lastname: Szerencsei
authors.forename: C F
authors.initials: CF
authors.lastname: Prinsen
authors.forename: P P
authors.initials: PP
authors.lastname: Schnetkamp
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002132:Calcium Radioisotopes / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002645:Chickens; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry / Q000378:metabolism*; D012414:Rubidium Radioisotopes / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism
chemical_list: D002132:Calcium Radioisotopes; D002352:Carrier Proteins; D012414:Rubidium Radioisotopes; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D012964:Sodium; D002118:Calcium