Apparent affinity of the Na/K pump for ouabain in cultured chick cardiac myocytes: Effects of Nai and K0

Abstract

The measured apparent affinity (K0.5) of the Na/K pump for ouabain has been reported to vary over a wide range. In a previous report we found that changing Nai could alter apparent affinity by at least an order of magnitude and that the model presented predicted this variability. To increase our understanding of this variability, isolated cells or two- to three-cell clusters of cardiac myocytes from 11-d embryonic chick were used to measure the effects of Nai and Ko on the K0.5 of the Na/K pump for ouabain. Myocytes were whole-cell patch clamped and Na/K pump current (Ip) was measured in preparations exposed to a Ca-free modified Hank's solution (HBSS) that contained 1 mM Ba, 10 mM Cs, and 0.1 mM Cd. Under these conditions there are no Ko-sensitive currents other than Ip because removal of Ko in the presence of ouabain had no effect on the current-voltage (I-V) relation. The I-V relation for Ip showed that in the presence of 5.4 mM Ko and 51 mM Nai, Ip has a slight voltage dependence, decreasing ∼30% from 0 to -130 mV. Increasing Nai in the patch pipette from 6 to 51 mM (Ko = 5.4 mM) caused Ip to increase from 0.46 ± 0.07 (n = 5) to 1.34 ± 0.08 μA/cm2 (n = 13) with a K0.5 for Nai of 17.4 mM and decreased the K0.5 for ouabain from 18.5 ± 1.8 (n = 4) to 3.1 ± 0.4 μM (n = 3). Similarly, varying Ko between 0.3 and 10.8 mM (Nai = 24 mM) increased Ip from 0.13 ± 0.01 (n = 5) to 0.90 ± 0.05 μA/cm2 (n = 5) with a K0.5 for Ko of 1.94 mM and increased K0.5 for ouabain from 0.56 ± 0.14 (n = 3-6) to 10.0 ± 1.1 μM (n = 6). All of these changes are predicted by the model presented. A qualitative explanation of these results is that Nai and Ko interact with the Na/K pump to shift the steady-state distribution of the Na/K pump molecules among the kinetic states. This shift in state distribution alters the probability that the Na/K pump will be in the conformation that binds ouabain with high affinity, thus altering the apparent affinity. In intact cells, the measured apparent affinity represents a combination of all the rate constants in the model and does not equate to simple first-order binding kinetics. The results show that the apparent affinity for ouabain is variable (0.5-20 μM) and depends on Nai and Ko. Although this range is large, the data can be completely accounted for by the model presented.