2 FUNCTIONALLY DIFFERENT NA K PUMPS IN CARDIAC VENTRICULAR MYOCYTES/

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracel lular Naf is similar to 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0 .26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our mai n conclusion is the total Na/K pump current comprises currents from tw o different types of pumps, whose functional responses to the extracel lular environment are different. Pump current was reversibly blocked w ith two affinities by extracellular dihydro-ouabain (DHO). We determin ed dissociation constants of 72 mu M for low affinity (type-1) pumps a nd 0.75 mu M for high affinity (type-h) pumps. These dissociation cons tants did not detectably change with two intracellular Na+ concentrati ons, one saturating and one near half-saturating, and with two extrace llular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pump s were therefore measured using 5 mu M DHO and on total pump current u sing 1 mM DHO. Extracellular K+ half-maximally activated the type-h pu mps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the t ype-1 pumps with half-maximal blockade at a pH of 7.71 whereas the typ e-h pumps were insensitive to extracellular pH. Both types of pumps re sponded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below , had any effect on either type of pump. The lack of any effect of int racellular K+ suggests the dissociation constants are in the molar ran ge so this step in the pump cycle is not rate limiting under normal ph ysiological conditions. Changes in intracellular-Na+ did not affect th e half-maximal activation by extracellular K+, and vice versa. We foun d DHO-blockade of Na/K pump current in canine ventricular myocytes als o occurred with two affinities, which are very similar to those from g uinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular Kt activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracel lular pH and K+ are important determinants of Na/K pump current. (c) N ormal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K p ump current appears to be specifically tailored for a tissue by expres sion of a mix of functionally different types of pumps.