| The retinal rod Na/Ca+K exchanger couples the extrusion of Ca to the inward Na and outward K gradients, and rapidly lowers cytosolic Ca in the rod outer segment, providing a key cellular signal for the recovery from photoexcitation. At the start of this doctoral project, only the bovine rod Na/Ca+K exchanger had been cloned, and no information was available on structure function relationships.;In this dissertation I report the cloning of the second Na/Ca+K exchanger cDNA, the human rod NCKX1, as well as the determination of the associated chromosomal localization and genomic organization. Comparison of the cDNA sequences reveals unexpectedly low homology between the bovine and human homologues in the extracellular and cytoplasmic loop regions. However, the hydrophobic membrane-spanning domains show strong conservation. Examination of the genomic organization revealed that the human rod NCKX1 exchanger contains nine introns, five of them clustered in a short region that codes for the N-terminal portion of the cytosolic loop. Analysis of human and bovine retinal transcripts found that alternate splicing occurs in the cytoplasmic region where the gene is divided by the multiple introns. The human rod NCKX1 gene was localized to chromosome 15q22-23, and ongoing investigations are looking into the role of NCKX1 in human retinopathy.;Construction and heterologous expression of deletion and chimeric bovine Na/Ca+K exchanger mutants demonstrated that only the two large hydrophobic transmembrane segments are required for Nai-dependent Ca influx, ie. reverse exchange. In addition, the Na/Ca+K exchanger characteristic K-dependence is maintained in the heterologously expressed deletion mutant, as measured by Km activation constants for Ca and K. |
