Functional expression of Na–Ca exchanger clones measured with the fluorescent Ca2+-indicating dye fluo-3

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Abstract
The process of Ca2+homeostasis is of prime importance to all cells because of the ubiquitous role of cytoplasmic Ca2+as an intracellular messenger and the cytotoxicity of sustained elevated cytosolic Ca2+concentrations. Two classes of plasma membrane proteins are responsible for 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 exchangers are known, the 3Na:1Ca exchangers found in heart, brain, kidney, and most other tissues and the 4Na:1Ca+1K exchanger found in retinal rod and cone photoreceptors. Functional expression of Na–Ca(/K) exchangers is most often measured as45Ca uptake in Na+-loaded cells or as Na–Ca exchange currents with the giant excised patch technique. In this study, two functional assays used to detect expression of the bovine heart Na–Ca exchanger in CHO cells are described. Both assays are based 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.Key words: Na–Ca exchange, Ca homeostasis, fluo-3, ion transport, functional expression.