Effect of Ki on the K, Dependence of Na/K Pump Current in Adult Rat Cardiac Myocytesa T. A. KINARD AND J. R. STIMERSb Department of Pharmacology and Toxicology University of Arkansas for Medical Sciences Little Rock, Arkansas 72205 Our laboratory group is in the process of investigating how ions interact in regulating Na/K pump function and how these interactions affect the Na/K pump’s sensitivity to cardiac glycosides. Previously, we used the whole-cell patch-clamp technique to examine the effect of internal “a] (Na,) on the external [K] (K,,) dependence of the Na/K pump current (I,) in adult rat ventricular myocytes and found a Hill coefficient (n) of approximately 1 (FIG. 1, B).’ To explore why n was not 2 as predicted by models of the Na/K pump, the effect of internal [K] (Ki) was evaluated by altering the pipette [K] (Kp) and keeping the pipette “a] (Na,) constant at 30 mM (total monovalent cation concentration was 160 mM). Adult rat ventricular myocytes were isolated by digestion of Langendorf€ perfused hearts with collagenase.2 The wholecell patch-clamp technique was used to measure I, in myocytes voltage clamped at -40 mV and maintained at 3PC. The external test solutions contained 1mM Ba, 0.1 mM Cd, and 0 Ca to block Ca and K currents to improve the isolation of I,. Using a protocol described previ~usly,~ the pump was initially deactivated in a K-free solution and then reactivated by brief applications of solutions containing K, ranging between 0.17 and 10.8 mM. The K, dependence of I, was measured as the change in holding current from baseline to the initial peak produced when I, was reactivated by brief applications of K, solutions. Currents were normalized to cell membrane capacitance to get pump current density and then were curve fitted with the Hill equation. The best fit parameter values for each data set are given in TABLE 1 Our initial hypothesis was that when & = 75 mM and K, = 0 mM, K, leaking out of the cell accumulated in the transverse tubules (t-tubules) and partially reactivated the Na/K pump. This reactivation then caused an underestimation of n in our experiment. To test this, &was replaced with an equimolar substitution of Cs,. This resulted in an increase of n from 1.1 to 1.3 (FIG. 1, 0 ; TABLEl), supporting the hypothesis; however, n was still lower than expected. As Cs is a Kmimic on the Na/K pump, it is possible that the high Cs, (130 rnM) might also be affecting our results. We therefore substituted tetramethylammonium (TMA) for Cs, which resulted in an almost twofold increase in maximal I, (Ipmax), but little change in the Hill coefficient (FIG. 1,O; TABLE 1). The 47% reduction in Ipmax in the presence of Cs internally can be explained by Cs inhibiting the maximum turnover rate of the Na/K pump by favoring the reverse reaction. However, the decrease (rather than increase) in n, although unexpected, may be complicated by the increase in Ipmax.It is possible that aThis work was supported by NIH Grants HL44660 and RR06564. bTowhom correspondence should be addressed. 458

KINARD & STIMERS Na/K PUMP

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459

c

2.0

-

1.5

-

1.0

-

0.5

-

0 1

.

0

1

,

2

4

.

I

6

.

,

I

8

10

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KO(mM) FIGURE 1. Summary of the data (means 2 SEM) obtained from adult rat ventricular cells when measuring the K, dependence of I,. The internal solutions contained 30 mM Na, 75 mM K and 55 mM Cs (W), 130 mM Cs ( O ) ,130 mM TMA ( O ) ,or 130 mM TMA 10 )IM ouabain (0).Thesolid curves represent the best fit of the Hill equation to each data set.

+

in addition to the accumulation of K in the t-tubules when K, is low and I, small, the high turnover rate when K,, is high causes a depletion of K from the t-tubules. To investigate if the high turnover rate with T M A internal solution caused a K depletion from the t-tubules, 10 p,M ouabain was added to the external solutions to reduce Ipma by approximately 50% (FIG. 1, 0). Decreasing I, with ouabain resulted in an l), suggesting that K, depletion increase in the Hill coefficient from 1.2 to 1.4 (TABLE from the t-tubules may be occurring in these experiments and cause an underestimation of n. When similar experiments were performed on embryonic chick cardiac myocytes, which do not have t-tubules, a Hill coefficient of 1.7 was found.3 This suggests that the t-tubules in rat ventricular myocytes may interfere with I, measurements when K, is limiting. In summary, these data suggest that (1) Ki leaking out of the cell may accumulate in the t-tubules and partially reactivate the pump, ( 2 ) Cs binding to the K, binding sites inhibits the maximum turnover rate of the Na/K pump, and (3) K, depletion from the t-tubules may occur when pump activity is high. Each of these effects may cause a distortion when measuring K, dependence of the Na/K pump activity.

TABLE1. Parameter Values for Hill Equation Internal Solutions 75 mM K, 55 mM Cs 130 mM Cs 130 mM TMA 130 mM TMA + 10 )IM ouabain

1.5 1.6 3.0 1.6

2.6 2.2 2.4 1.8

1.1 1.3 1.2 1.J

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ANNALS NEW YORK ACADEMY OF SCIENCES REFERENCES

1. KINARD,T.A. & J. R. STIMERS. 1991. Effect of Nai on the K, dependence of Na/K pump current in adult rat cardiac myocytes. Biophys. J. 61: A406 2. STIMERS, J. R. 1992. Extracellular perfusion controller for electrophysiology. J. Neurosci. Methods 43:139-151. 3. STIMERS, J. R., N. SHIGETO & M. LIEBERMAN. 1990. Na/K pump current in aggregates of cultured chick cardiac myocytes. J. Gen. Physiol. 9 5 61-76.