Overview: stoichiometry and voltage dependence of the Na/K pump.
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Electric Conductivity↗
Biomedical subjects
D C Gadsby
Publications and source records attributed to D C Gadsby.
Influence of Na/K pump current on action potentials in Purkinje fibers.
Moderate changes in the size of the outward (hyperpolarizing) current that is generated directly by the electrogenic Na/K exchange pump in the surface membrane of cardiac Purkinje fibers can cause substantial alterations in the shape of the action potential, in the level of the diastolic potential, or of the resting potential of quiescent cells, or in the rate of firing of spontaneously active preparations. Transient increments in Na/K pump current, of suitable magnitude, can be elicited experimentally in small canine Purkinje fibers by causing a transient increase in their intracellular Na concentration, [Na]i, and, thereby, a transient increase in the rate of electrogenic Na extrusion. Two techniques were used to increase [Na]i: in the first, the rate of Na extrusion from the cells was temporarily reduced by omitting K ions from the bathing fluid for short periods of time, in the second, the rate of Na entry into the cells was temporarily increased by electrically stimulating the preparations rapidly (e.g., greater than or equal to 2 Hz) for brief periods. After the extracellular K concentration was restored, or after electrical stimulation was stopped, respectively, use of a two-microelectrode voltage-clamp technique allowed the resulting increments in pump current to be measured directly, as changes in holding current. Increments in pump current elicited by these two methods in the same preparation decline with the same exponential time-course. In preparations stimulated electrically at a regular, low rate (e.g., less than or equal to 1 Hz) both methods of temporarily stimulating the Na/K pump cause a marked, transient reduction in the duration of the action potential. A closely similar reduction in action-potential duration to that observed during enhanced pump activity can be elicited by injecting, from an external source, a steady hyperpolarizing current of magnitude similar to that of the increment in pump current recorded in the same preparation under voltage clamp.
Action Potentials↗
The Na/K pump of cardiac cells.
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Action Potentials↗
Influence of the sodium pump current on electrical activity of cardiac cells.
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Animals↗
The basis for the membrane potential of quiescent cells of the canine coronary sinus.
During prolonged periods of quiescence, the membrane potential of cells in the isolated canine coronary sinus, exposed to normal Tyrode solution containing 4 mM-K, declines to about -60 mV. The nature of the resting potential was investigated, in small strips of coronary sinus tissue mounted in a fast-flow system, by recording the membrane potential responses to sudden changes in the extracellular ionic environment. At extracellular K concentrations ([K]o) from 0 to 64 mM the resting potential was little affected by replacing all but 1 mM of external Cl ions with isethionate and methylsulphate ions. At [K]o levels from 4 to 150 mM the resting potential was reasonably well described by the Goldman-Hodgkin-Katz equation on the assumption that the intracellular K concentration ([K]i) was 155 mM and that the ratio of membrane permeability coefficients for Na and K, PNa/PK, was 0.07. In the presence of a high concentration of acetylcholine or carbachol (greater than or equal to 1 microM), the resting potentials at [K]o levels from 1 to 150 mM approximated K equilibrium potentials (EK) calculated on the assumption that [K]i was 155 mM. At [K]o levels less than or equal to 8 mM replacing most of the external Na with sucrose or Tris caused a substantial hyperpolarization, whereas application of 1-2 microM-tetrodotoxin caused only slight hyperpolarization. A transient hyperpolarization, due to enhanced electrogenic Na extrusion, was recorded on switching back to 4 mM-K following brief exposures to K-free solution; no transient hyperpolarization was recorded in the presence of 5 microM-acetylstrophanthidin. The acetylstrophanthidin itself caused a rapid depolarization of several millivolts. Preliminary conductance measurements made with two micro-electrodes in some smaller preparations indicate that the steady-state current-voltage relationship is N-shaped. We conclude that the low membrane potential of quiescent coronary sinus cells reflects not a low [K]i but rather a relatively high ratio PNa/PK, of about 0.07: the Na ions flow into the cells via predominantly TTX-insensitive pathways and are extruded by the electrogenic Na/K exchange pump, which thereby makes a substantial contribution to the resting potential.
Acetylcholine↗
Noradrenaline hyperpolarizes cells of the canine coronary sinus by increasing their permeability to potassium ions.
The mechanism of the noradrenaline-induced hyperpolarization was investigated in small strips of coronary sinus tissue mounted in a fast-flow system. The recorded hyperpolarization was negligibly small in response to 10 nM-noradrenaline but was maximal at 10 microM (average amplitude 23 mV, in 4 mM-K solution). The hyperpolarization was unaffected by 1 microM-phentolamine but was abolished by 10 microM-propranolol and so is presumably mediated via beta-adrenoceptors. The noradrenaline-induced hyperpolarization became smaller when the extracellular K concentration ([K]o) was raised or when the extracellular Na concentration was lowered. These results are consistent with two general mechanisms: noradrenaline might cause hyperpolarization by stimulating the Na/K pump to generate more outward current, as previously suggested for other cell types. Alternatively, noradrenaline might lower the permeability ratio, PNa/PK, by reducing the permeability coefficient for Na (PNa) and/or increasing that for K (PK). The noradrenaline-induced hyperpolarization is not diminished during exposure to 5 microM-acetylstrophanthidin, or to K-free solution, or to K-free solution containing acetylstrophanthidin. We conclude that the hyperpolarization does not reflect enhanced electrogenic pump activity. Conductance measurements using two micro-electrodes in very small preparations revealed that, like the muscarinic agonist carbachol, noradrenaline caused an increase in membrane slope conductance. Steady-state current-voltage curves obtained in the presence of noradrenaline, in the presence of carbachol, and in the absence of both drugs all crossed each other at about the same level of membrane potential. During the maintained injection of sufficiently large hyperpolarizing current, application of either noradrenaline or carbachol causes depolarization instead of hyperpolarization. The cross-over or 'reversal' potentials of current-voltage curves, determined with and without the drugs, vary with [K]o approximately as does the K equilibrium potential calculated assuming the intracellular K concentration to be 155 mM. We conclude that, like carbachol and acetylcholine, noradrenaline causes a specific increase in the K permeability of coronary sinus cells.
Animals↗
Electrogenic sodium extrusion can stop triggered activity in the canine coronary sinus.
Soon after a burst of triggered activity in the canine coronary sinus begins, an initial fall in maximum diastolic potential and increase in rate gives way to an increase in maximum diastolic potential, reduction in rate, and eventual quiescence. This hyperpolarization, slowing, and subsequent quiescence might result from enhanced electrogenic sodium/potassium extrusion caused by the rise in intracellular sodium concentration ([Na+]i) associated with the high rate of firing. Triggered bursts can be terminated prematurely by a sudden increase in the rate of sodium extrusion, Brief exposure to K+- free fluid is known to cause [Na+]i to rise; reactivating the pump by switching back to K+-containing fluid causes immediate hyperpolarization, and within a few seconds, quiescence. Brief periods of overdrive, also thought to increase [Na+]i, are followed by hyperpolarization, slowing and, often, by premature termination of the burst. Inhibiting the sodium/potassium pump by exposure to 2 micrometer acetylstrophanthidin or to K+-free fluid (1) prevents or delays the hyperpolarization, (2) increases the rate of triggered activity and (3) prolongs bursts of activity when bursts last less than 2.5 minutes under control conditions. In the presence of 2 micrometer acetylstrophanthidine, neither brief exposures to K+- free fluid not overdrive causes sudden, premature termination of triggered bursts. Bursts do eventually stop in the presence of pump inhibitors; however, that termination is associated with an increase in rate and a decline in maximum diastolic potential and in action potential amplitude. We conclude that electrogenic Na+ extrusion plays an important role in the spontaneous termination of triggered activity.
Animals↗
Activation of electrogenic Na+/K+ exchange by extracellular K+ in canine cardiac Purkinje fibers.
Transient increments in sodium pump current were elicited in small voltage-clamped Purkinje fibers suspended in a fast flow system by briefly exposing them to K+-free fluid, to temporarily inhibit the pump, and then suddenly returning them to K+-containing fluid. The exponential time course of decay of the current increment provides a measure of the pump rate constant for Na+ extrusion. The dependence of that rate constant, and of the peak amplitude of the increment in pump current, on the extracellular K+ concentration was determined. The results indicate: that in cardiac Purkinje cells, as in many other cells, the pump is half-maximally activated by about 1 mM K+; that the coupling ratio for Na+/K+ exchange is independent of either intracellular Na+ concentration or external K+ concentration; and that a simple model in which intracellular Na+ concentration is determined by a passive "leak," and an active extrusion of Na+, seems sufficient to account for moderate changes in cellular Na+ concentration.
Animals↗
Direct measurement of changes in sodium pump current in canine cardiac Purkinje fibers.
Purkinje fibers from dog hearts may have either a "high" resting potential of about -90 mV or a "low" resting potential of about -40 mV when immersed in low-Cl(-) solution containing 4 mM K(+). Brief exposure of Purkinje fibers at the low level of resting potential to K(+)-free fluid causes further depolarization, and return to K(+)-containing solution elicits a transient hyperpolarization which reaches a peak within a few seconds and then declines within a few minutes. Repeating these changes in K(+) concentration after clamping the membrane potential at its steady resting level in K(+)-containing fluid allows the changes in net membrane current presumably underlying the depolarization and transient hyperpolarization to be measured. Net inward current is recorded when the fiber is exposed to K(+)-free solution, and a transient net outward current arises when it is returned to K(+)-containing solution. The transient net outward current reflects a temporary increase in the rate of electrogenic Na(+) extrusion caused by the rise in intracellular Na(+) concentration that occurs while the sodium pump is slowed in K(+)-free fluid. Sodium extrusion remains enhanced, presumably until the internal Na(+) concentration has been brought back to its resting level. The transient outward current is completely abolished by the cardiac steroid acetylstrophanthidin, and its amplitude is increased as the prior exposure to K(+)-free fluid is prolonged. The decay of the transient outward current and the decline in intracellular Na(+) concentration both appear to follow first-order kinetics.
Animals↗
Electrogenic sodium extrusion in cardiac Purkinje fibers.
Thin canine cardiac Purkinje fibers in a fast flow chamber were exposed to K-free fluid for 15 s to 6 min to initiate "sodium loading," then returned to K-containing fluid to stimulate the sodium pump. The electrophysiological effects of enhanced pump activity may result from extracellular K depletion caused by enhanced cellular uptake of K or from an increase in the current generated as a result of unequal pumped movements of Na and K, or from both. The effects of pump stimulation were therefore studied under three conditions in which lowering the external K concentration ([K]0) causes changes opposite to those expected from an increase in pump current. First, the resting potential of Purkinje fibers may have either a "high" value of a "low" (less negative) value: at the low level of potential, experimental reduction of [K]0 causes depolarization, whereas an increase in pump current should cause hyperpolarization. Second, in regularly stimulated Purkinje fibers, lowering [K]0 prolongs the action potential, whereas an increase in outward pump current should shorten it. Finally, lowering [K]0 enhances spontaneous "pacemaker" activity in Purkinje fibers, whereas an increase in outward pump current should reduce or abolish spontaneous activity. Under all three conditions, we find that the effects of temporary stimulation of the sodium pump are those expected from a transient increase in outward pump current, not those expected from K depletion.
Action Potentials↗
The effects of acetylcholine on the electrical activity of canine cardiac Purkinje fibers.
We studied the effects of acetylcholine (ACh) on small bundles of canine cardiac Purkinje fibers exposed to normal, or low-chloride (isethionate) Tyrode's solution in a rapid superfusion system. In superfusate containing 4 mM K+, the resting potential of Purkinje fibers may be either "low," near -40 mV, or "high," near -90 mV. ACh, at 10(-6) to 10 (-5) M, increased the membrane potential from both the low and high resting levels and, in low-Cl solution, often induced a maintained shift in potential from the low to the high level. The increase in membrane potential caused by ACh was greater at the low than at the high level. ACh, at 10(-6) to 10(-5) M, reduced action potential duration in both normal and low-Cl Tyrode's solution, the effect being more marked in the latter. These effects of ACh were reversibly abolished by atropine (5 X 10(-5) M), indicating that they were mediated via muscarinic ACh receptors, and they probably result from an increase in membrane K+ conductance since 10(-5) M ACh reversibly reduced, by 13% on the average, the amplitudes of the steady changes in membrane potential evoked by applying small current pulses (-5 to -25 nA, 200 msec). ACh (10(-5) M) also diminished the rate of, or stopped, spontaneous activity arising from either level of membrane potential. The cessation of spontaneous slow response activity, arising from the low level, sometimes was accompanied by a maintained shift of the membrane potential to the high resting level. It is concluded that the action of ACh on Purkinje fibers is qualitatively similar to its action on sinoatrial nodal and atrial cells.
Acetylcholine↗
The dual nature of the membrane potential increase associated with the activity of the sodium/potassium exchange pump in skeletal muscle fibres.
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Animals↗
Two levels of resting potential in cardiac Purkinje fibers.
In an appropriate ionic environment, the resting potential of canine cardiac purkinje fibers may have either of two value. By changing the external K concentration, [K](0), in small steps, it was shown that, in the low (1 mM) Cl, Na-containing solutions used in this study, the two levels of resting potential could be obtained only within a narrow range of [K](0) values; that range was usually found between 1 and 4 mM. Within the critical [K](0) range the resting potential could be shifted from either level to the other by the application of small current pulses. It was shown that under these conditions the steady-state current- voltage relationship was "N-shaped," and that a region of both negative slope, and negative chord conductance lay between the two stable zero-current potentials. The negative chord conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord conductance could be abolished by several experimental interventions and the membrane potential thereby shifted from the lower to the higher resting level: those interventions which were effective by presumably diminishing the steady-state inward current included reducing the external sodium concentration, adding TTX, or adding lidocaine; those which presumably increased the steady-state outward current included small increases in [K](0), brief depolarizations to around -20 mV, or the addition of acetylcholine chloride.
Action Potentials↗
Do intracellular concentrations of potassium or sodium regulate the strength of the heart beat?
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Animals↗
Beta-adrenoceptor agonists increase membrane K+ conductance in cardiac Purkinje fibres.
Hormonal modulation of the ionic conductance of cell membranes is a topic of considerable current interest; it has a major role, for example, in the improved performance of the vertebrate heart elicited by sympathetic nerve stimulation or by circulating catecholamines, an effect involving enhanced calcium influx. beta-Agonist catecholamines also abbreviate the action potential of cardiac Purkinje fibres, and increase the resting potential in a variety of cells, including cardiac cells, a hyperpolarization usually attributed to stimulation of the electrogenic Na+/K+ pump. We show here that nanomolar concentrations of beta-catecholamines cause hyperpolarization of cardiac Purkinje fibres, not by increasing Na+/K+ pump current, but by increasing resting membrane K+ conductance. The hyperpolarization and shortening of the action potential should increase availability of Na+ channels and reduce the refractory period, effects tending to safeguard impulse propagation through the ventricular conducting system despite the increased heart rate caused by beta-catecholamine action on the sinus node pacemaker.
Adrenergic beta-Agonists↗
Voltage dependence of Na/K pump current in isolated heart cells.
The Na/K pump usually pumps more Na+ out of the cell than K+ in, and so generates an outward component of membrane current which, in the heart, can be an important modulator of the frequency and shape of the cardiac impulse. Because it is electrogenic, Na/K pump activity ought to be sensitive to membrane potential, and it should decline with hyperpolarization. However, such voltage dependence of outward pump current has yet to be demonstrated, one reason being the technical difficulty of accurately measuring pump current over a sufficiently wide voltage range. The whole-cell patch-clamp technique allows effective control of both intracellular and extracellular solutions as well as membrane voltage. Applying this technique to myocardial cells isolated from guinea pig ventricle, we have measured Na/K pump current between -140 mV and +60 mV, after minimizing passive currents flowing through Ca2+, K+ and Na+ channels. We report here that strongly activated pump current shows marked voltage dependence; it declines steadily from a maximal level near 0 mV, becoming very small at -140 mV. Pump current-voltage relationships will provide essential information for testing models of the Na/K pump mechanism and for predicting pump-mediated changes in the electrical activity of excitable cells.
Animals↗
Voltage dependence of Na translocation by the Na/K pump.
During each complete reaction cycle, the Na/K pump transports three Na ions out across the cell membrane and two K ions in. The resulting net extrusion of positive charge generates outward membrane current but, until now, it was unclear how that net charge movement occurs. Reasonable possibilities included a single positive charge moving outwards during Na translocation; or a single negative charge moving inwards during K translocation; or either positive or negative charges moving during both translocation steps, but in unequal quantities. Any step that involves net charge movement through the membrane must have voltage-dependent transition rates. Here we report measurements of transient, voltage-dependent, displacement currents generated by the pump when its normal Na/K transport cycle has been interrupted by removal of external K and it is thus constrained to carry out Na/Na exchange. The quantity and voltage sensitivity of the charge moved during these transient currents suggests that Na translocation includes a voltage-dependent transition involving movement of one positive charge across the membrane. This single step can thus fully account for the electrogenic nature of Na/K exchange. The result provides important new insight into the molecular mechanism of active cation transport.
Animals↗