The Na/K pump maintains the electrochemical gradients for Na and K ions across the surface membrane of most mammalian cells. The pump extrudes about 3 Na ions for every 3 K ions taken up, and so generates an outward (hyperpolarizing) component of membrane current which, in the heart, can modulate the frequency and shape of the cardiac impluse. Na/K pump current also provides a suitable signal for monitoring pump rate. Because the pump is electrogenic, its rate under certain conditions is expected to be voltage-dependent and should decline with hyperpolarization. We have demonstrated such voltage dependence of pump current. Most available kinetic information on the Na/K pump, however, has been obtained without regard for membrane voltage, usually near zero membrane potential. The research is aimed to fill this gap and determine the influence of membrane potential on the transport properties of the Na/K pump by measuring pump current-voltage relationships over a wide range of membrane potentials (-140 to +60 mV) and under a wide variety of ionic and metabolic conditions. Cells isolated from guinea pig ventricles by collagenase treatment will be studied using the whole-cell patch-clamp technique, in combination with wide-tipped patch pipettes and a device for changing the solution within the pipette, so that the compositions of both intracellular and extracellular solutions, as well as membrane potential, can be controlled effectively. Solutions are designed to minimize passive currents flowing through Ca, K, and Na ion channels while sustaining Na/K pump current. Effects of different levels of intracellular and extracellular [Na] and [K], and of changes in intracellular [ATP], [ADP], and [Pi], will be examined systematically. Na/K pump current-voltage relationships will be determined as the difference between current-voltage relationships obtained in the absence and in the presence of a cardiotonic steroid such as strophanthidin. The relative shapes, amplitudes and positions along the voltage axis of the pump current-voltage relationships under these various conditions will provide kinetic and thermodynamic information essential for developing and testing mechanistic models of the Na/K pump. Because changes in Na/K pump current can markedly alter the electrical activity of cardiac cells, and probably also of nerve cells, such detailed pump current-voltage relationships should also allow more accurate interpretation, or simulation, of electrophysiological consequences of changes in pump rate.
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