MgATP counteracts intracellular proton inhibition of the sodium-calcium exchanger in dialysed squid axons

Abstract

Intracellular Na(+) and H(+) inhibit Na(+)-Ca(2+) exchange. ATP regulates exchange activity by altering kinetic parameters for Ca(2+)(i), Na(+)(i) and Na(+)(o). The role of the Ca(2+)(i)regulatory site on Na(+)(i)-H(+)(i)-ATP interactions was explored by measuring the Na(+)(o)-dependent (45)Ca(2+) efflux (Na(+)(o)-Ca(2+)(i) exchange) and Ca(2+)(i)-dependent (22)Na(+) efflux (Na(+)(o)-Na(+)(i) exchange) in intracellular-dialysed squid axons. Our results show that: (1) without ATP, inhibition by Na(+)(i) is strongly dependent on H(+)(i). Lowering the pH(i) by 0.4 units from its physiological value of 7.3 causes 80 % inhibition of Na(+)(o)-Ca(2+)(i) exchange; (2) in the presence of MgATP, H(+)(i) and Na(+)(i) inhibition is markedly diminished; and (3) experiments on Na(+)(o)-Na(+)(i) exchange indicate that the drastic changes in the Na(+)(i)-H(+)(i)-ATP interactions take place at the Ca(2+)(i) regulatory site. The increase in Ca(2+)(i) affinity induced by ATP at acid pH (6.9) can be mimicked by a rise in pH(i) from 6.9 to 7.3 in the absence of the nucleotide. We conclude that ATP modulation of the Na(+)-Ca(2+) exchange occurs by protection from intracellular proton and sodium inhibition. These findings are predicted by a model where: (i) the binding of Ca(2+) to the regulatory site is essential for translocation but not for the binding of Na(+)(i) or Ca(2+)(i) to the transporting site; (ii) H(+)(i) competes with Ca(2+)(i) for the same form of the exchanger without an effect on the Ca(2+)(i) transporting site; (iii) protonation of the carrier increases the apparent affinity and changes the cooperativity for Na(+)(i) binding; and (iv) ATP prevents both H(+)(i) and Na(+)(i)-effects. The relief of H(+) and Na(+) inhibition induced by ATP could be important in cardiac ischaemia, in which a combination of acidosis and rise in [Na(+)](i) occurs.

Figure 1. Effect of intracellular protons on the forward Na⁺-Ca²⁺ exchange in the absence of Nai+ and ATP

Ca²⁺ efflux in the presence (•) and absence (○) of ${\text{Na}}_{\text{o}}^{+}$. Note the strong inhibition by protons of the forward Na⁺-Ca²⁺ exchange. Axon diameter, 650 μm. Temperature, 17.5 °C.

Figure 2. Cai2+-dependent activation of forward Na⁺-Ca²⁺ exchange flux at different values of pH_i in the absence of Nai+ and ATP

A, ${\text{Na}}_{\text{o}}^{+}$-dependent Ca²⁺ efflux at different values of [Ca²⁺]_i at pH 6.9 (•), 7.3 (○), 7.7 (▾) and 8.8 (▿). The error bars indicate s.e.m. The mean temperature was 17 °C. B, fractional inhibition by ${\text{H}}_{\text{i}}^{+}$ expressed as the ratio of the forward Na⁺-Ca²⁺ exchange at pH 6.9 relative to that at pH 7.3, 7.7 and 8.8 as a function of [Ca²⁺]_i. The graph was constructed using data from the experiments shown in Fig. 2A.

Figure 9

A, cartoon showing ligand interactions between ${\text{Na}}_{\text{i}}^{+}$-${\text{H}}_{\text{i}}^{+}$ (competition-synergism) and the protective effect of ATP on these interactions. NCX, Na⁺-Ca²⁺ exchanger; R, regulatory site; and T, transport site. B, state diagram of the Na⁺-Ca²⁺ exchanger. See text for detaspan

Figure 3. Effect of Nai+ on the forward Na⁺-Ca²⁺ exchange at different values of pH_i in the absence of ATP

${\text{Na}}_{\text{o}}^{+}$-dependent Ca²⁺ efflux at pH 6.9 (A) 7.3 (B) and 8.8 (C) in the presence (•) and absence (○) of ${\text{Na}}_{\text{o}}^{+}$. All concentrations are millimolar. Notice the marked synergism between ${\text{Na}}_{\text{i}}^{+}$ and ${\text{H}}_{\text{i}}^{+}$ in inhibiting the exchanger at acidic pH_i. D, ${\text{Na}}_{\text{i}}^{+}$-dependent inhibition of forward Na⁺-Ca²⁺ exchange at different values of pH_i in the absence of ATP. Ordinate, percentage ${\text{Na}}_{\text{o}}^{+}$-dependent Ca²⁺ efflux (${\text{Na}}_{\text{o}}^{+}$-${\text{Ca}}_{\text{i}}^{2+}$ exchange). The error bars indicate s.e.m. The mean temperature was 17 °C. Notice the exquisite sensitivity of the exchange activity to ${\text{Na}}_{\text{i}}^{+}$ at the acidic pH.

Figure 4. Effect of ATP on forward Na⁺-Ca²⁺ exchange in the presence and absence of Nai+ at different values of intracellular pH_i

A, ${\text{Na}}_{\text{o}}^{+}$-dependent Ca²⁺ efflux in the absence of ${\text{Na}}_{\text{i}}^{+}$ in the presence (•) and absence (○) of ${\text{Na}}_{\text{o}}^{+}$. Notice the small effect of ATP at pH 7.3 compared to the large activation at pH 6.9. Axon diameter, 525 μm. B, ${\text{Na}}_{\text{o}}^{+}$-dependent Ca²⁺ efflux in the presence of a physiological [Na⁺]_i of 40 mm in the presence (•) and absence (○) of ${\text{Na}}_{\text{o}}^{+}$. Observe the large effect of ATP at pH 7.3 and its miniscule effect at pH 8.8. Axon diameter, 620 μm. C, ${\text{H}}_{\text{i}}^{+}$-dependent inhibition of forward Na⁺-Ca²⁺ exchange at physiological ${\text{Na}}_{\text{i}}^{+}$ (40 mm) in the absence (•) and presence (○) of ATP (3 mm). The error bars indicate s.e.m. The mean temperature was 17 °C. Notice that the major fractional activation of the Na⁺-Ca²⁺ exchange by ATP occurs between pH 6.9 and 7.3.

Figure 5. ATP relief of Nai+-Hi+ inhibition of forward Na⁺-Ca²⁺ exchange

A, ${\text{Na}}_{\text{i}}^{+}$-induced inhibition of ${\text{Na}}_{\text{o}}^{+}$-dependent Ca²⁺ efflux at pH 6.9 in an axon dialysed first without ATP, then with 3 mm ATP. Notice first, the large activation in the exchange activity induced by ATP in the absence of ${\text{Na}}_{\text{i}}^{+}$ and second, the relief of ${\text{Na}}_{\text{i}}^{+}$ inhibition. B, ${\text{Na}}_{\text{i}}^{+}$-dependent inhibition of forward Na⁺-Ca²⁺ exchange at pH 6.9 in the presence and absence of ATP. C, ${\text{Na}}_{\text{i}}^{+}$-dependent inhibition of forward Na⁺-Ca²⁺ exchange at pH 8.8 with and without ATP. The error bars indicate s.e.m.

Figure 6. Effect of acid and alkaline pH_i on the Ca^2*_i-dependent Nao+-Nai+ exchange

Steady-state ${\text{Na}}_{\text{o}}^{+}$-dependent Na⁺ efflux at pH 6.9 induced by increasing the [Ca²⁺]_I from 0 to 200 μm (A) and steady-state ${\text{Na}}_{\text{o}}^{+}$-dependent Na⁺ efflux at pH 8.8 induced by increasing [Ca²⁺]_i from 0 to 10 μm (B) in the presence (•)and absence (○) of ${\text{Na}}_{\text{o}}^{+}$. The [Ca²⁺]_i was controlled with dibromoBAPTA (see Methods). Notice the large change in the apparent affinity of the ${\text{Na}}_{\text{o}}^{+}$-${\text{Na}}_{\text{i}}^{+}$ exchange for ${\text{Ca}}_{\text{i}}^{2+}$ between acid and alkaline pH. C, percentage ${\text{Ca}}_{\text{i}}^{2+}$-dependent ${\text{Na}}_{\text{o}}^{+}$-${\text{Na}}_{\text{i}}^{+}$ exchange at pH 6.9 (○), 7.3 (▾) and 8.8 (•) in the absence of ATP at a physiological [Na⁺]_i. The measurements at 0.3 μm ${\text{Ca}}_{\text{i}}^{2+}$ were obtained with BAPTA as Ca²⁺ chelator. All other measurements were carried out with dibromoBAPTA. The error bars indicate s.e.m.D, percentage ${\text{Ca}}_{\text{i}}^{2+}$-dependent ${\text{Na}}_{\text{o}}^{+}$-${\text{Na}}_{\text{i}}^{+}$ exchange at pH 6.9 in the presence (•) and absence (○) of 3 mm ATP. The error bars indicate s.e.m. The mean temperature was 17.5 °C.

Figure 7. The effect of pCMBS on the alkalinization-induced increase in Nao+-Cai2+ and Nao+-Nai+ exchange in axons dialysed without ATP

${\text{Na}}_{\text{o}}^{+}$-dependent Na⁺ efflux (A) and ${\text{Na}}_{\text{o}}^{+}$-dependent Ca²⁺ efflux (B) in the presence (•) and absence (○) of ${\text{Na}}_{\text{o}}^{+}$. The numbers above the lines represent the buffered intracellular pH. The arrows indicate the addition of 1 mmpCMBS. Notice that pCMBS completely blocks the alkalinization-induced increase in both ${\text{Na}}_{\text{o}}^{+}$-${\text{Ca}}_{\text{i}}^{2+}$ and ${\text{Na}}_{\text{o}}^{+}$-${\text{Na}}_{\text{i}}^{+}$ exchange. Notice also that at the end of both experiments increasing ${\text{Ca}}_{\text{i}}^{2+}$ to saturating values reactivates the fluxes to normal levels. The mean temperature was 17.5 °C.

Figure 8. Kinetic model simulations for the Nai+, Hi+, Cai2+ and ATP interactions in the regulation of the squid Na⁺-Ca²⁺ exchanger

Notice that with the values of the constant used, at physiological pH, ${\text{Ca}}_{\text{i}}^{2+}$ and ${\text{Na}}_{\text{i}}^{+}$, the fraction of carriers available for translocation is quite small. See text for details.