Kinetic comparisons of heart and kidney Na+,K(+)-ATPases

Abstract

Most kinetic measurements of the partial reactions of Na(+),K(+)-ATPase have been conducted on enzyme from mammalian kidney. Here we present a kinetic model that is based on the available equilibrium and kinetic parameters of purified kidney enzyme, and allows predictions of its steady-state turnover and pump current in intact cells as a function of ion and ATP concentrations and the membrane voltage. Using this model, we calculated the expected dependence of the pump current on voltage and extracellular Na(+) concentration. The simulations indicate a lower voltage dependence at negative potentials of the kidney enzyme in comparison with heart muscle Na(+),K(+)-ATPase, in agreement with experimental results. The voltage dependence is enhanced at high extracellular Na(+) concentrations. This effect can be explained by a voltage-dependent depopulation of extracellular K(+) ion binding sites on the E2P state and an increase in the proportion of enzyme in the E1P(Na(+))(3) state in the steady state. This causes a decrease in the effective rate constant for occlusion of K(+) by the E2P state and hence a drop in turnover. Around a membrane potential of zero, negligible voltage dependence is observed because the voltage-independent E2(K(+))(2) → E1 + 2K(+) transition is the major rate-determining step.

Figure 1

Simplified representation of the Albers-Post scheme describing the Na⁺,K⁺-ATPase catalytic cycle. Step 1: Binding of three Na⁺ ions from the cytoplasm, phosphorylation by ATP, and occlusion of Na⁺ within the protein. Step 2: Conformational change of the phosphorylated protein releasing the Na⁺ ions to the extracellular medium. Step 3: Binding of two K⁺ ions from the extracellular medium, occlusion of K⁺ within the protein, and dephosphorylation. Step 4: Conformational change of the unphosphorylated protein releasing K⁺ ions to the cytoplasm.

Figure 2

Scheme describing the coupled equilibria of Na⁺ and K⁺ binding to the E2P conformation of the enzyme. The model assumes competition between Na⁺ and K⁺ at two of the transport sites with dissociation constants K_N and K_K, respectively, and one specific Na⁺ transport site with a dissociation constant K_N1.

Figure 3

Dependence of the Na⁺,K⁺-ATPase current-voltage relationship (I/V curve) on the extracellular Na⁺ concentration. Symbols correspond to the following Na⁺ concentrations: 1.5 mM (○), 50 mM (●), 100 mM (□), and 150 mM (■). The solid lines between the points have simply been drawn to aid the eye of the reader. Upper curve: Experimental results for guinea pig heart ventricular myocytes, obtained via the whole-cell patch-clamp technique, reproduced from Fig. 3 of Nakao and Gadsby (19). The pump current, I_p, of each curve has been normalized to the value obtained at a holding potential of + 40 mV. The experimental conditions were [Na⁺]_cyt = 50 mM, [K⁺]_cyt = 0 mM, [K⁺]_ext = 5.4 mM, [ATP]_cyt = 10 mM, T = 36°C. Lower curve: Computer simulations of the expected I/V curve for mammalian kidney Na⁺,K⁺-ATPase pump current based on the kinetic and equilibrium parameters given in Table 1 and the Albers-Post scheme described by Figs. 1 and 2. The ion concentrations, ATP concentration, and temperature used for the simulations were the same as for the upper curve.

Figure 4

Simulation of a voltage-jump transient. The initial conditions were [Na⁺]_cyt = 80 mM, [Na⁺]_ext = 25 mM, [K⁺]_cyt = [K⁺]_ext = 0 mM, [ATP]_cyt = 5 mM, V_m = −120 mV, T = 22°C, as in the experiments of Gadsby et al. (30). At time = 0, the membrane voltage was jumped to 0 mV. The current transient is attributed to the voltage-dependent release of Na⁺ from the E2P state, which is rate-limited by the E1P(Na⁺)₃ → E2PNa⁺₃ transition.

Figure 5

Simulations of the time courses of the relative populations of the Na⁺,K⁺-ATPase in the conformational states E1, E2, E1P, and E2P after a jump in the cytoplasmic Na⁺ concentration from 15 to 150 mM. All other conditions ([Na⁺]_ext = 140 mM, [K⁺]_cyt = 120 mM, [K⁺]_ext = 4 mM, [ATP]_cyt = 3 mM, V_m = −80 mV, T = 24°C) were held constant. The simulations were based on the kinetic and equilibrium parameters given in Table 1.