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+] 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 (INaCa) using patch clamp methods. INaCa 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 INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa 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 INaCa, 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.
Egger
M
M
Department of Physiology, University of Bern, Switzerland.
Ruknudin
A
A
Lipp
P
P
Kofuji
P
P
Lederer
W J
WJ
Schulze
D H
DH
Niggli
E
E
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D009206:Myocardium / Q000378:metabolism; D009532:Nickel / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; D009532:Nickel; D002118:Calcium