FUNCTIONAL EXPRESSION OF NA-CA EXCHANGER CLONES MEASURED WITH THE FLUORESCENT CA2-INDICATING DYE FLUO-3()

The process of Ca2+ homeostasis is of prime importance to all cells be cause of the ubiquitous role of cytoplasmic Ca2+ as an intracellular m essenger and the cytotoxicity of sustained elevated cytosolic Ca2+ con centrations. Two classes of plasma membrane proteins are responsible f or maintaining cytosolic free Ca2+ in the submicromolar range against a very large electrochemical Ca2+ gradient across the plasma membrane, the ATP-driven Ca2+ pump and Na-Ca exchangers. Two types of Na-Ca exc hangers are known, the 3Na:1Ca exchangers found in heart, brain, kidne y, and most other tissues and the 4Na:1Ca+1K exchanger found in retina l rod and cone photoreceptors. Functional expression of Na-Ca(/K) exch angers is most often measured as Ca-45 uptake in Na+-loaded cells or a s Na-Ca exchange currents with the giant excised patch technique. In t his study, two functional assays used to detect expression of the bovi ne heart Na-Ca exchanger in CHO cells are described. Both assays are b ased on measurements of cytosolic free Ca2+ with the fluorescent Ca2+- indicating dye fluo-3 and should be equally applicable in the study of functional expression of both Na-Ca and Na-Ca/K exchanger clones.