Abstract
Na,K -ATPase containing the amino acid substitution glutamate to alanine at position 779 of the α subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na
+
–Na
+
exchange activityin the absence of K
+
. In microsomal preparations of Glu779Ala enzyme, the Na
+
concentration for half maximal activation of Na-ATPase activity was 161 ± 14 mM (
n
= 3). Furthermore, enzyme activity with 800 mM Na
+
was found to be similar in the presence and absence of 20 mM K
+
. These results showed that Na
+
, with low affinity, could stimulate enzyme turnover as effectively as K
+
. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na
+
during superfusion in K
+
-free solutions. Electrogenic Na
+
–Na
+
exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na
+
(Na
+
o
) concentration. At all Na
+
o
concentrations tested (3–148 mM), exchange current was maximal at negative membrane potentials (
V
M
), but decreased as
V
M
became more positive. Analyzing this current at each
V
M
with a Hill equation showed that Na
+
–Na
+
exchange had a high-affinity, low-capacity component with an apparent Na
+
o
affinity at 0 mV (
K
0
0.5
) of 13.4 ± 0.6 mM and a low-affinity, high-capacity component with a
K
0
0.5
of 120 ± 13 mM (
n
= 17). Both high- and low-affinity exchange components were
V
M
dependent, dissipating 30 ± 3% and 82 ± 6% (
n
= 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na
+
–Na
+
exchange could be explained by Na
+
o
acting as a low-affinity K
+
congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na
+
o
binding at a Na
+
-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K -ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000.
J. Gen. Physiol
. 116:47–59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K -ATPase.