Altered Na + transport after an intracellular α-subunit deletion reveals strict external sequential release of Na + from the Na/K pump

The Na/K pump actively exports 3 Na+ in exchange for 2 K+ across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the α-subunit diminishes internal Na+ (Nai + ) affinity ≈25-fold [Morth et al. (2007)Nature 450:1043–1049]. Because external Na+ (Nao + ) binding is voltage-dependent, we studied the reactions involving this process by using two-electrode and inside-out patch voltage clamp in normal and truncated (ΔKESYY) Xenopus-α1 pumps expressed in oocytes. We observed that ΔKESYY (i ) decreased both Nao + and Nai + apparent affinities in the absence of Ko + , and (ii ) did not affect apparent Nao + affinity at high Ko + . These results support a model of strict sequential external release of Na+ ions, where the Na+ -exclusive site releases Na+ before the sites shared with K+ and the ΔKESYY deletion only reduces Nao + affinity at the shared sites. Moreover, at nonsaturating Ko + , ΔKESYY induced an inward flow of Na+ through Na/K pumps at negative potentials. Guanidinium+ can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidiniumo + can also traverse normal Na/K pumps in the absence of both Nao + and Ko + and can also inhibit Na/K pump currents in a Na+ -like voltage-dependent manner, we conclude that the normal pathway transited by the first externally released Na+ is large enough to accommodate guanidinium+ .