| The Na+, K+-ATPase is a primary active transporter that transports three Na+ out of the cell and two K+ into the cell. The pump produces a net charge transfer, and is therefore electrogenic, so the generated current can serve as a measure of the pump activity. In order to test the current-voltage (I-V) relationship of the sodium pump, Bufo marinus Na+ , K+-ATPase alpha and beta subunits were cloned and their functional properties were tested using a Xenopus laevis expression system and two-electrode voltage clamp techniques. The steady state pump current is known to be voltage dependent (Sagar and Rakowski, 1994), and can be activated by extracellular K+, and inhibited by ouabain, which is a specific inhibitor of the sodium pump. We determined the ouabain-sensitive current as a measure of the pump-mediated current with different concentrations of ouabain used to inhibit the exogenous K+ activated pump current. The voltage dependence of the ouabain-sensitive pump current was shown to be due to the voltage dependence of the sodium pump turnover rate, because the inhibition of pump current by ouabain did not depend on membrane potential. The Ki value for ouabain inhibition is 30 +/- 3 muM. Pump current activated by addition of K+ was not equal to the current inhibited by ouabain, contrary to results previously obtained that measured the endogenous Xenopus pump current using the compound tetraethylammonium (TEA+). TEA+ has been widely used to block K + channels and permit measurement of pump current simply by addition of external K+. A series of experiments was perfomed in the absence of external K+ to test if TEA+ is itself capable of activating Bufo pump current. In both high (100 mM) and Na+-free external conditions, TEA+ elicited K+-like activation of the ouabain sensitive current. TEA + activates an electrogenic pump current with a lower affinity than that of K+.;The X-ray crystal structure of pig renal Na+, K +-ATPase at 3.5 A resolution has been determined with two occluded rubidium ions. These two sites are expected to be occupied by Na+ and K+ alternatively. The position of the third Na+ binding site is still hypothetical. Mutations were used to examine the effects of changing the candidate amino acids of the binding site. These mutations change the voltage dependence and apparent affinity of extracellular Na+ binding. These results suggest that the third Na + binding site is located between the fifth, sixth, and ninth transmembrane helices of the Na+, K+-ATPase alpha subunit. |
