Functional expression of the human cardiac Na⁺/Ca²⁺ exchanger in Sf9 cells: Rapid and specific Ni²⁺ transport

Although inhibition of the Na⁺/Ca²⁺ exchanger normally increases [Ca²⁺](i) in neonatal cardiac myocytes, application of the inhibitor Ni²⁺ appears to reduce [Ca²⁺](i) measured by fluo-3. To investigate how the apparent reduction in [Ca²⁺](i) occurs we examined Ca²⁺ transport by the human Na⁺/Ca²⁺ exchanger expressed in Sf9 cells. Transport of Ca²⁺ by the Na⁺/Ca²⁺ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca²⁺ indicator fluo-3, and the electrogenic function was determined by measuring the Na⁺/Ca²⁺ exchange current (I(NaCa)) using patch clamp methods. I(NaCa) was elicited with voltage-clamp steps or flash photolysis of caged Ca²⁺. We show significant expression of Na⁺/Ca²⁺ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na⁺/Ca²⁺ exchanger. In addition to measurements of I(NaCa), characterization includes Ca²⁺ transport via the Na⁺/Ca²⁺ exchanger and the voltage dependence of Ca²⁺ transport. Application of Ni²⁺ blocked I(NaCa) but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni²⁺ was transported via the Na⁺/Ca²⁺ exchanger at a rate comparable to the Ca²⁺ transport. Once inside the cells, Ni²⁺ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni²⁺ does indeed block I(NaCa), but is also rapidly translocated across the cell membrane by the Na⁺/Ca²⁺ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.