Na pump current can be separated into ouabain-sensitive and -insensitive components in single rat ventricular myocytes.
The present study was undertaken to identify functional isoforms of the Na,K-ATPase in single rat cardiac myocytes. Na,K-ATPase activity was measured as ouabain-sensitive, extracellular K-activated outward current (Na pump current) in ventricular myocytes voltage-clamped with single low-resistance (0.5- 1 M omega) patch electrodes at 36 degrees C. Solutions to block contaminating currents allowed Na pump current to be measured without significant contamination in 140 mM Na-containing superfusion solutions. The current-voltage relationship had a positive slope at potentials from -125 to 0 mV but became almost voltage-independent at positive potentials. The apparent Km for activation of this current at -40 mV by extracellular K was 2.7 +/- 0.3 mM (mean +/- SEM, n = 3) and increasing electrode Na increased the amplitude of the current to a maximum density of 4.11 +/- 0.17 pA/pF (n = 34). Intracellular vanadate (100 microM) produced an extracellular K-dependent inhibition of Na pump current that was rapidly reversed in K-free superfusion solution. Dose-dependent inhibition of Na pump current by ouabain was best described as the sum of two Michaelis-Menten binding sites: one with higher affinity (K1/2 = 1.0 +/- 0.7 microM) comprising 33 +/- 9% (n = 5-6) of the total current and the second with a K1/2 of 43 +/- 14 microM. Changing electrode [Na] from 15 to 100 mM had no effect on the dose-dependent inhibition of the current by ouabain. Thus, the properties of high and low affinity components of Na pump current are consistent with the presence of different Na,K-ATPases isoforms that have a similar ion dependence for transport activity.
Bockus Research Institute, Graduate Hospital, Philadelphia PA 19146, USA.
N
N
Ishizuka
A J
AJ
Fielding
J R
JR
Berlin
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D006352:Heart Ventricles; D007425:Intracellular Membranes / Q000378:metabolism; D007536:Isomerism; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000502:physiology*; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D014638:Vanadates; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium