Stoichiometry of Na+-Ca2+ exchange is 3:1 in guinea-pig ventricular myocytes

Abstract

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.

Figure 1. E_NCX at 1 and 2 mm [Ca²⁺]_o at three different HPs

A, concatenated current responses without pulse intervals. [Ca²⁺]_o was raised from 1 to 2 mm at −73 mV HP. Ni²⁺ was applied to block I_NCX and detect E_NCX at each [Ca²⁺]_o. B, I-V curves of control (a) and in the presence of Ni²⁺ (b) at 1 mm [Ca²⁺]_o obtained from A. C, I-V curves of control (c) and in the presence of Ni²⁺ (d) at 2 mm [Ca²⁺]_o obtained from A. D, I-V curves of net Ni²⁺-sensitive currents obtained by subtraction from B (a - b) and C (c - d). E, I-V curves of net Ni²⁺-sensitive currents obtained by subtraction at 1 and 2 mm [Ca²⁺]_o at −42 mV HP. F, I-V curves of net Ni²⁺-sensitive currents at 1 and 2 mm [Ca²⁺]_o at −11 mV HP.

Figure 2. Effect of raising [Na⁺]_o on E_NCX at three different HPs

A, concatenated current responses without pulse intervals. [Na⁺]_o was raised from 140 to 200 mm at −73 mV HP. B, I-V curves of control (a) and in the presence of Ni²⁺ (b) at 140 mm [Na⁺]_o. C, I-V curves of control (c) and in the presence of Ni²⁺ (d) at 200 mm [Ca²⁺]_o. D, I-V curves of net Ni²⁺-sensitive currents obtained by subtraction from B (a - b) and C (c - d). E, subtracted I-V curves of Ni²⁺-sensitive currents at 140 and 200 mm [Na⁺]_o at −42 mV HP. F, subtracted I-V curves of Ni²⁺-sensitive currents at 140 and 200 mm [Na⁺]_o at −11 mV HP.

Figure 4. Effects of −90 mV HP (E_NCX at a 3:1 stoichiometry) and −20 mV HP (E_NCX at a 4:1 stoichiometry) on E_NCX during the recovery from Ni²⁺ inhibition

A, concatenated current responses at −90 mV HP. B, concatenated current responses at −20 mV HP. C, difference I-V curves of the corresponding labels from A. E_NCX were around −90 mV and did not dramatically change with time. D, difference I-V curves of the corresponding labels from B. E_NCX was initially at about −70 mV and shifted toward −20 mV HP with time.

Figure 5. Effects of −20 mV HP (E_NCX at a 3:1 stoichiometry) and −90 mV HPs on E_NCX during the recovery of I_NCX from Ni²⁺ inhibition

A, concatenated currents at −20 mm HP which is E_NCX at a 3:1 stoichiometry. B, concatenated currents at −90 mV HP which is a more negative voltage than E_NCX at a 3:1 stoichiometry. C, difference I-V curves of the corresponding labels from A. E_NCX was at about −20 mV and did not shift with time. D, difference I-V curves of the corresponding labels from B. E_NCX developed initially at around −20 mV and then shifted toward −34 mV with time.

Figure 3. [Ca²⁺]_i measurement with indo-1 at two different HPs

[Ca²⁺]_i was measured using a confocal microscopy with 100 μm indo-1 in the pipette under the voltage-clamp. A, at −11 mV HP, [Ca²⁺]_i was 374 ± 38 nm (n = 16) (left). B, in the same cell at −73 mV HP, [Ca²⁺]_i was 173 ± 11 nm (n = 13). (P < 0.0002 by Student's t test).