Voltage Dependence of the Apparent Affinity for External Na+ of the Backward-Running Sodium Pump

Abstract

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, Ipump (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 (Ipump-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various Vm to H2DTG or from the difference between membrane I-V relationships obtained by imposing Vm 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 Ipump-V relationship. The reverse Ipump-V relationship is sigmoid, Ipump saturating at large negative Vm, 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 = nλ, where n (1.33 ± 0.05) is the Hill coefficient of Na binding, and λ (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.