Stoichiometry of Na+-Ca2+ exchange is 3:1 in guinea-pig ventricular myocytes.
In single guinea-pig ventricular myocytes, we examined the stoichiometry of Na(+)-Ca(2+) exchange (NCX) by measuring the reversal potential (E(NCX)) of NCX current (I(NCX)) and intracellular Ca(2+) concentration ([Ca(2+)](i)) with the whole-cell voltage-clamp technique and confocal microscopy, respectively. With given ionic concentrations in the external and pipette solutions, the predicted E(NCX) were -73 and -11 mV at 3:1 and 4:1 stoichiometries, respectively. E(NCX) measured were -69 +/- 2 mV (n = 11), -47 +/- 1 mV (n = 14) and -15 +/- 1 mV (n = 15) at holding potentials (HP) of -73, -42 and -11 mV, respectively. Thus, E(NCX) almost coincided with HP, indicating that [Ca(2+)](i) and/or [Na(+)](i) changed due to I(NCX) flow. Shifts of E(NCX) (deltaE(NCX)) were measured by changing [Ca(2+)](o) or [Na(+)](o). The measured values of deltaE(NCX) were almost always smaller than those expected theoretically at a stoichiometry of either 3:1 or 4:1. Using indo-1 fluorescence, [Ca(2+)](i) measured under the whole-cell voltage-clamp supported a 3:1 but not 4:1 stoichiometry. To prevent Ca(2+) accumulation, we inhibited I(NCX) with Ni(2+) and re-examined E(NCX) during washing out Ni(2+). With HP at predicted E(NCX) at a 3:1 stoichiometry, E(NCX) developed was close to predicted E(NCX) and did not change with time. However, with HP at predicted E(NCX) for a 4:1 stoichiometry, E(NCX) developed initially near a predicted E(NCX) for a 3:1 stoichiometry and shifted toward E(NCX) for a 4:1 stoichiometry with time. We conclude that the stoichiometry of cardiac NCX is 3:1.
Hinata
Masamitsu
M
Department of Pharmacology, School of Medicine, School of Nursing, Fukushima Medical University, Fukushima 960-1295, Japan. mhinata@fmu.ac.jp
Yamamura
Hisao
H
Li
Libing
L
Watanabe
Yasuhide
Y
Watano
Tomokazu
T
Imaizumi
Yuji
Y
Kimura
Junko
J
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D004594:Electrophysiology; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D066298:In Vitro Techniques; D007211:Indoles; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D009206:Myocardium / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D009532:Nickel / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000502:physiology*
D005456:Fluorescent Dyes; D007211:Indoles; D019831:Sodium-Calcium Exchanger; D009532:Nickel; D012964:Sodium; C048960:indo-1; D002118:Calcium