Functional Characterization and Molecular Cloning of the K+-dependent Na+/Ca2+ Exchanger in Intact Retinal Cone Photoreceptors

Light-dependent changes in cytoplasmic free Ca2+ are much faster in the outer segment of cone than rod photoreceptors in the vertebrate retina. In the limit, this rate is determined by the activity of an electrogenic Na+/Ca2+ exchanger located in the outer segment plasma membrane. We investigate the functional properties of the exchanger activity in intact, single cone photoreceptors isolated from striped bass retina. Exchanger function is characterized through analysis both of the electrogenic exchanger current and cytoplasmic free Ca2+ measured with optical probes. The exchanger in cones is K+ dependent and operates both in forward and reverse modes. In the reverse mode, the K+ dependence of the exchanger is described by binding to a single site with K1/2 about 3.6 mM. From the retina of the fish we cloned exchanger molecules bassNCKX1 and bassNCKX2. BassNCKX1 is a single class of molecules, homologous to exchangers previously cloned from mammalian rods. BassNCKX2 exists in four splice variants that differ from each other by small sequence differences in the single, large cytoplasmic loop characteristic of these molecules. We used RT-PCR (reverse transcriptase polymerase chain reaction) of individual cells to identify the exchanger molecule specifically expressed in bass single and twin cone photoreceptors. Each and every one of the four bassNCKX2 splice variants is expressed in both single and twin cones indistinguishably. BassNCKX1 is not expressed in cones and, by exclusion, it is likely to be an exchanger expressed in rods