Stoichiometry of the retinal cone Na/Ca-K exchanger heterologously expressed in insect cells: Comparison with the bovine heart Na/Ca exchanger

The transport stoichiometry is an essential property of antiporter and symp orter transport proteins. In this study, we determined the transport stoich iometry of the retinal cone potassium-dependent Na/Ca exchanger (NCKX) expr essed in sodium-loaded cultured insect cells. The Na/Ca and Rb/Ca coupling ratios were obtained by direct measurements of the levels of Rb-86 and Ca-4 5 uptake and sodium release associated with reverse Na/Ca exchange. Rb/Ca c oupling ratios of 0.98 [standard deviation (SD) of 0.12, 15 observations] a nd 0.92 (SD of 0.12, 13 observations) were obtained for the chicken and hum an 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 f or the chicken and human retinal cone NCKX, respectively, whereas a lower a verage coupling ratio of 3.11 (SD of 0.34, 10 observations) was obtained wi th cells expressing the bovine Na/Ca exchanger (NCX1). These results are co nsistent 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 remove d all showed a significant calcium-dependent 86Rb uptake, whereas no calciu m-dependent 86Rb uptake was observed in cells expressing bovine NCX1. The c alcium dependence of 45Ca uptake yielded values between 1 and 2.5 muM for t he external calcium dissociation constant of the different NCKX proteins st udied here.