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Original Article

^a Marine Biological Laboratory, Woods Hole, Massachusetts 02543

deweer{at}mail.med.upenn.edu

The steady-state voltage and [Na⁺]_o dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K⁺-free seawater; ATP- and Na⁺-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated ⁴²K⁺ efflux to reverse pump current, I_pump (both defined by sensitivity to dihydrodigitoxigenin, H₂DTG), scaled by Faraday's constant, was –1.5 ± 0.4 (n = 5; expected ratio for 2 K⁺/3 Na⁺ stoichiometry is –2.0). Steady-state reverse pump current-voltage (I_pump-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V_m to H₂DTG or from the difference between membrane I-V relationships obtained by imposing V_m staircases in the presence or absence of H₂DTG. With the second method, we also investigated the influence of [Na⁺]_o (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I_pump-V relationship. The reverse I_pump-V relationship is sigmoid, I_pump saturating at large negative V_m, and each doubling of [Na⁺]_o causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state ²²Na⁺ efflux during electroneutral Na⁺/Na⁺ exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100–103). This model predicts valence z = n, where n (1.33 ± 0.05) is the Hill coefficient of Na binding, and (0.61 ± 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na⁺/Na⁺ exchange modes only with additional assumptions that render them less likely.

Key Words: Na,K-ATPase • electrogenicity • active transport • kinetics • modeling

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