Voltage dependence of the apparent affinity for external Na+ of the backward-running sodium pump

The steady-state voltage and [Na+](0) 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 K-42(+) efflux to reverse pump current, I-pump (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), 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 with from the shifts in holding current after repeated exposures of an axon clamped at various Vm to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing Vm staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na+](0) (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, Ipump saturating at large negative Vm, and each doubling of [Na+](0) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltxmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state Na-22(+) 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 lambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding site. More elaborate alternative models can accomodate all the steady-state features of the reverse pumping and electroneutral Na+/Na+ exchange modes only with additional assumptions that render them less likely.