Charge movements via the cardiac Na,K-ATPase.
The voltage dependence of transient and steady-state pump currents was examined in guinea pig ventricular myocytes to investigate mechanisms of charge translocation by the Na,K-ATPase. Na/K pump current was determined at approximately 36 degrees C as strophanthidin-sensitive whole-cell current in myocytes voltage clamped and internally dialyzed via wide tipped pipettes containing a pipette perfusion device. External Na ions diminished stationary pump current during forward Na/K cycling in a voltage dependent manner, the inhibition becoming stronger upon hyperpolarization. When Na,K-ATPase activity was restricted to Na translocation steps, stationary pump current was prevented but voltage pulses still elicited large transient pump currents which could be abolished by oligomycin B (> or = 2 micrograms/ml). The transients arose instantaneously on stepping the voltage, and decayed with voltage-dependent approximately single exponential time courses. The decay rates, and their high temperature sensitivity (approximately 200 s-1 at 0 mV at 36 degrees C; approximately 40 s-1 at 20 degrees C), suggest that the charge movements were limited by a conformational change associated with Na deocclusion. Those rates varied asymmetrically with voltage, changing little at positive voltages but increasing roughly exponentially with hyperpolarization (e-fold/approximately 80 mV). Lowering the extracellular [Na] ([Na]o) slowed the relaxation of charge movement at negative potentials but had little effect at positive potentials, and so shifted the rate constant-voltage curve to the left. The implied dependence on [Na]o of the backward rate constant governing pump charge movement accounts satisfactorily for the observed [Na]o sensitivity of stationary outward Na/K pump current, and indicates that the voltage-dependent step somehow involves the release of Na ions to the external medium. However, no strophanthidin-sensitive current was seen, at saturating external [K], when Na,K-ATPase activity was limited to K translocation steps by complete withdrawal of Na ions. But, at very low [Na]o, a weak negative slope appeared in the stationary pump current-voltage relationship at subsaturating, but not at saturating, external [K], indicating an increased apparent affinity for external K at more negative potentials. The results support the existence of a high field access channel through which extracellular Na and K ions must pass before interacting with their binding sites deep within the Na,K-ATPase molecule.
Gadsby
D C
DC
Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, New York 10021.
Nakao
M
M
Bahinski
A
A
Nagel
G
G
Suenson
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004594:Electrophysiology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007474:Ion Exchange; D007700:Kinetics; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D015221:Potassium Channels / Q000502:physiology; D015222:Sodium Channels / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism* / Q000502:physiology
D015221:Potassium Channels; D015222:Sodium Channels; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase