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

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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 Na~ 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 Na~ and K o on the K05 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 K o and 51 mM Na~, Ip has a slight voltage dependence, decreasing ~ 30% from 0 to - 130 mV. Increasing Na~ in the patch pipette from 6 to 51 mM (K o -- 5.4 mM) caused Ip to increase from 0.46 _ 0.07 (n = 5) to 1.34 -+ 0.08 ~/cm 2 (n = 13) with a K05 for Na i of 17.4 mM and decreased the K05 for ouabain from 18.5 _+ 1.8 (n = 4) to 3.1 _+ 0.4 ~M (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 txA/cm 2 (n = 5) with a K05 for Ko of 1.94 mM and increased K05 for ouabain from 0.56 - 0.14 (n = 3-6) to 10.0 - 1.1 I~M (n = 6). All of these changes are predicted by the model presented. A qualitative explanation of these results is that Na~ and Ko interact with the Na/K pump to shift the steady-state distribution of the Na/K pump molecules among the

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