AJP - Cell Physiology, Vol 253, Issue 1 C126-C136, Copyright © 1987 by American Physiological Society
Measurement of Na+-K+ coupling ratio of Na+-K+-ATPase in rabbit proximal tubules
M. J. Avison, S. R. Gullans, T. Ogino, G. Giebisch and R. G. Shulman
A combination of 23Na nuclear magnetic resonance (NMR) spectroscopy and a
K+-selective electrode was used to make simultaneous measurements of net
Na+ and K+ fluxes across plasma membranes of rabbit renal proximal tubules
after an abrupt stimulation of Na+-K+-ATPase. After a step in extracellular
K+ concentration ([K+]o) from low to higher concentration (0.1-0.3 mM to
0.5-5.2 mM) at 25 degrees C, net extrusion of Na+ and uptake of K+ were
observed. These fluxes were completely inhibited by ouabain (10(-3) M).
Because initial rates of K+ uptake in presence or absence of Ba2+ (a known
inhibitor of plasma membrane K+ conductance) were indistinguishable, net K+
flux was virtually unidirectional. Because suspension buffers contained
neither glucose nor amino acids and the ratio of net Na+ and K+ fluxes (JNa
and JK, respectively) was constant over a wide range of transmembrane Na+
gradients and absolute values of the JNa and JK, it is likely that changes
in electrogenic or passive net fluxes across plasma membranes were
insignificant in the first 30-45 s after the [K+]o step. Thus the ratio of
these initial net Na+ and K+ fluxes corresponds closely to the Na+-K+
coupling ratio of the Na+-K+-ATPase. In 12 experiments, the measured
Na+-K+-ATPase coupling ratio was 1.54 +/- 0.07 (SE). The coupling ratio was
constant over a wide range of intracellular Na+ content, intracellular
sodium concentration, [K+]o and transmembrane Na+ gradient. The coupling
ratio also remained constant over an eightfold range of Na+-K+-ATPase
rates.
