Charge translocation by the Na⁺/K⁺ pump under Na⁺/Na⁺ exchange conditions: intracellular Na⁺ dependence

¹ NINDS
² Ohio University

^* To whom correspondence should be addressed. E-mail: rakowski{at}ohio.edu.

Submitted on August 22, 2005
Revised on September 14, 2005
Accepted on 14 November 2005

	Abstract

ABSTRACT The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40 ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the -sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four state kinetic model describing the Na+ binding, occlusion, conformational change and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved the mid-point voltage of the charge distribution (Vq) shifted -25.3 ± 0.4 mV which can be accounted for by the presence of an extracellular ion well having a dielectric distance = 0.69 ± 0.01. The effect of changes of on -sensitive transient current was investigated. The mid-point voltage (Vq) of the charge distribution curve was not affected over the concentration range 3.13 to 50 mM. As was decreased the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2 ± 0.2 mM. The effects of lowering on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.

Key Words: ABC transporter, Na,K-ATPase, Xenopus oocyte, active transport, charge movement