Effects of reduced electrochemical Na+ gradient on contractility in skeletal muscle: role of the Na+-K+ pump

 Continued excitation of skeletal muscle may induce a combination of a low extracellular Na⁺ concentration ([Na⁺]_o) and a high extracellular K⁺ concentration ([K⁺]_o) in the T-tubular lumen, which may contribute to fatigue. Here, we examine the role of the Na⁺-K⁺ pump in the maintenance of contractility in isolated rat soleus muscles when the Na⁺, K⁺ gradients have been altered. When [Na⁺]_o is lowered to 25 mM by substituting Na⁺ with choline, tetanic force is decreased to 30% of the control level after 60 min. Subsequent stimulation of the Na⁺-K⁺ pump with insulin or catecholamines induces a decrease in [Na⁺]_i and hyperpolarization. This is associated with a force recovery to 80–90% of the control level which can be abolished by ouabain. This force recovery depends on hyperpolarization and is correlated to the decrease in [Na⁺]_i (r = 0.93; P<0.001). The inhibitory effect of a low [Na⁺]_o on force development is considerably potentiated by increasing [K⁺]_o. Again, stimulation of the Na⁺-K⁺ pump leads to rapid force recovery. The Na⁺-K⁺ pump has a large potential for rapid compensation of the excitation-induced rundown of Na⁺, K⁺ gradients and contributes, via its electrogenic effect, to the membrane potential. We conclude that these actions of the Na⁺-K⁺ pump are essential for the maintenance of excitability and contractile force. Received: 19 December 1996 / Received after revision: 25 March 1997 / Accepted: 2 April 1997     

Internal K ions modulate the action of external cations on hyperpolarization-activated inward current in rabbit isolated sinoatrial node cells

We have investigated the effect of change in external Na⁺ concentration on the hyperpolarization-activated inward current (I _f) in the presence of different internal cations. Rabbit single isolated sinoatrial node cells were studied using the whole-cell patch-clamp technique. With 140 mM K⁺ pipettes, lowering [Na⁺]₀ causes the fully activated I/V curve for I _f to shift in a negative direction without a significant decrease of the slope conductance. The P _Na/P _K ratio, as defined by the Goldman-Hodgkin-Katz equation, is concentration-dependent: the lower the [Na⁺]_o, the higher P _Na/P _K. The conductance/concentration relationship for I _f shows saturation at low [Na⁺]_o or [K⁺]_o, indicating that the channel has a strong affinity for external cations. With 140 mM Cs⁺ pipettes, the I/V curve shows strong inward rectification and inward I _f current decreases almost proportionally to the decrease in [Na⁺]_o; the conductance/ concentration relationship for I _f shifts to the right suggesting that the binding affinity of the external binding site is reduced. These results suggest that the I _f channel is a multi-ion channel with a high-affinity external binding site, the affinity of which is modulated by internal cations.     

Na+-activated K+ current in cardiac cells: rectification,open probability,block and role in digitalis toxicity

The Na⁺-activated K⁺ current was studied in inside-out patches and in whole cells isolated from the guinea-pig cardiac ventricle. The single channel conductance showed inward rectification for K⁺ _i+ _e, but outward rectification for K⁺ _i>K⁺ _e The open probability was dependent on Na⁺ _i and Na⁺,K⁺-pump activity. In the presence of pump blockade the channel remained active at low Na⁺ _i Similar results were obtained in whole cells. These results suggest the existence of Na⁺ gradients depending on Na⁺,K⁺-pump activity and passive inward leak of Na⁺. The channel and whole cell current were blocked by R56865. The drug did not change the single channel conductance but markedly reduced open probability by shortening burst duration. The current may play an important role in action potential shortening during pump blockade.This work was supported by a grant of the National Fund for Scientific Research Belgium.3.0016.87.     

Dependence of the electrogenic pump current of Xenopus oocytes on external potassium

The membrane potential of Xenopus oocytes showed a variable response to an increase of the K⁺ concentration in the bathing solution, [K⁺]_e, from 2.5 mM to 20 mM. In 54% of the cases (n=52) the cells hyperpolarized (by max. 70 mV). In the presence of 10^–5 M ouabain, all cells depolarized suggesting that the hyperpolarization was caused by an electrogenic Na⁺/K⁺ pump. In cells stored overnight in a Na⁺-free solution the transition from 2.5 to 20 mM [K⁺]_e always caused depolarization indicating that the stimulation of the pump requires high internal sodium, [Na⁺]_i. Cells stored overnight in a Na⁺-rich solution had a [Na⁺]_i of 30.7±7 mM, i.e. the Na⁺/K⁺ pump was saturated with sodium (Lafaire and Schwarz 1986). With 9 such cells we determined the K⁺ activation of the Na⁺/K⁺ pump. The activation follows Hill kinetics with I_max=90.5 nA, K_s=2.3 mM, and n=1.68.     

Role of external Ca2+ and K+ in gating of cardiac delayed rectifier K+ currents

We sought to determine whether extracellular Ca²⁺ (Ca _e ²⁺ ) and K⁺ (K _e ⁺ ) play essential roles in the normal functioning of cardiac K⁺ channels. Reports by others have shown that removal of Ca _e ²⁺ and K _e ⁺ alters the gating properties of neural delayed rectifier (I _K) and A-type K⁺ currents, resulting in a loss of normal cation selectivity and voltage-dependent gating. We found that removal of Ca _e ²⁺ and K _e ⁺ from the solution bathing guinea pig ventricular myocytes often induced a leak conductance, but did not affect the ionic selectivity or time-dependent activation and deactivation properties of I _K. The effect of [K⁺]_e on the magnitude of the two components of cardiac I _K was also examined. I _K in guinea pig myocytes is comprised of two distinct types of currents: I _Kr (rapidly activating, rectifying) and I _Ks (slowly activating). The differential effect of Ca _e ²⁺ on the two components of I _K (previously shown to shift the voltage dependence of activation of the two currents in opposite directions) was exploited to determine the role of K _e ⁺ on the magnitude of I _Ks and I _Kr. Lowering [K⁺]_e from 4 to 0 mM increased I _Ks, as expected from the change in driving force for K⁺, but decreased I _Kr. The differential effect of [K⁺]_e on the two components of cardiac I _K may explain the reported discrepancies regarding modulation of cardiac I _K conductance by this cation.     

Hyperpolarization slowly activates a potassium current in locust skeletal muscle

An inward current activated by hyperpolarization, I_K,H, was studied under voltage clamp in locust skeletal muscle. The dependence of its reversal potential on [K⁺]_o and its insensitivity to changes in [Na⁺]_o indicate that the underlying conductance is a K⁺ conductance. The instantaneous currentvoltage (I–V)-relationship exhibits outward rectification. Activation and deactivation take seconds and have complex time courses. At 10 mM [K⁺]_o activation seems to start at a voltage mV more positive than the resting potential ( –68 mV). Ba⁺⁺ blocks I_K,H strongly; so do Rb⁺ and Cs⁺, the latter in a voltage dependent manner. The slow inward current bears similarities to anomalous rectifiers as well as to mixed, hyperpolarization-activated K⁺/Na⁺ currents in other tissues.     

Ionic currents expressed in a cell line derived from the organ of Corti of the Immortomouse

 We have investigated the maturation of adult hair cell electrophysiology in a population of precursor cells in a conditionally immortal cell line. The cell line, UB/OC-2, from the embryonic organ of Corti of the H-2Kb-tsA58 transgenic mouse, permits cells to grow proliferatively at 33°C and to differentiate at 39°C. Whole-cell patch-clamp recordings showed that proliferating cells had a different electrophysiology to differentiating cells. Differentiating cells had a conditionally expressed slowly activating inward current activated by hyperpolarization. The current was not blocked by extracellular application of 0.5 mM Ba²⁺, but was blocked reversibly by 2 mM Cs⁺. The current was found to be carried by both K⁺ and Na⁺ ions (P _K/P _Na=2.2) and activated by 10 μM forskolin. These properties identify the slowly activating current as I _h. A proportion of proliferating and differentiating cells exhibited a voltage-gated Na⁺ current, I _Na. I _Na was abolished in Na-free external medium and was inhibited reversibly by tetrodotoxin (TTX) with K _i=64 nM. Together these results suggest that proliferating and differentiating hair cell precursors in the immortal cochlear cell line UB/OC-2 express currents which are also found in developing hair cells. Received: 6 January 1999 / Received after revision: 1 February 1999 / Accepted: 2 February 1999     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.     

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump

An increased extracellular K⁺ concentration ([K⁺]₀) is thought to cause muscle fatigue. We studied the effects of increasing [K⁺]₀ from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K⁺]₀ of 8–9 mM, a further small increase in [K⁺]₀ to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K⁺]_o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (E _M) relation showed only moderate force depression with depolarization from –74 to –62 mV, a large reduction of force occurred whenE _M fell to –53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na⁺-K⁺ pump with ouabain (10^–6 M) caused additional force loss at 11 mM [K⁺]₀. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na⁺-K⁺ pump in muscles exposed to 11 mM [K⁺ ₀] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofE _M (1.3 mV). The observations suggest that the increased Na⁺ concentration difference across the sarcolemma, following Na⁺-K⁺ pump stimulation, has an important role in restoring excitability and force.     

An early outward transient K+ current that depends on a preceding Na+ current and is enhanced by insulin

A whole-cell early transient outward current occurs in rat myoballs if and only if there is an immediatly preceding current of large amplitude through the voltage-gated, tetrodotoxin-inhibitable Na⁺ channel. This early outward transient is a K⁺ current, designated I _K(Na⁺). Under the conditions in which I _K(Na⁺) appears, simultaneous measurement of voltage and current, under voltage clamp, demonstrates that there is transient voltage escape to depolarized levels, peaking at about the time of peak inward Na⁺ current arid resembling an action potential. I _K(Na⁺) was never seen in the absence of this breach of the voltage clamp, suggesting that I _K(Na⁺) might be an artefact due to transient depolarization from the clamp. However, when the voltage escape was mimicked by voltage commands under conditions in which the Na⁺ channel was not activated, there was no I _K(Na). Insulin increased or produced I _K(Na⁺) even though insulin had no effect on I _Na or on the delayed rectifier K⁺ current or on the escape from voltage clamp. It is concluded that there is a population of rat myoballs in which there is an early outward K⁺ current that requires an immediately preceding current through the voltagegated tetrodotoxin-inhibitable Na⁺ channel and is enhanced by insulin.     

An analysis of the delayed outward current in single ventricular cells of the guinea-pig

Properties of the delayed outward current (I _K) in ventricular myocytes of the guinea-pig were studied using the whole cell clamp method. The experiments were performed under conditions in whichI _K was enhanced by application of isoproterenol while the Ca²⁺ current was eliminated by Ca²⁺-removal and by the addition of Cd²⁺. The reversal potential (E _rev) ofI _K, determined from the current tails, was about 10 mV less negative than the K⁺ equilibrium potential. This was estimated by examining the reversal potential of the inward rectifier K⁺ current in Ba²⁺-containing solution, or from the Nernst equation. TheE _rev-log[K⁺]₀ relationship had a slope of 49 mV per tenfold change in [K⁺]₀. In Na⁺-free solution,E _rev became more negative. Thus, although the major charge carriers inI _K are K⁺ ions, Na⁺ ions may also contribute in part to this current. TheP _Na/P _K ratio inI _K, calculated by applying a Goldman-Hodgkin-Katz relation to the reversal potential, was 0.016. The activation ofI _K during depolarization showed a sigmoidal time course at the onset, while the time course of the current tails was monoexponential at voltages more negative than –50 mV, but biexponential at more positive voltages. These observations can be explained by the conductance equation of the Hodgkin-Huxley type in which the kinetic variable is raised to the second power. These and other features ofI _K observed in the ventricular cells are discussed in comparison to the properties of similar current systems reported in other cardiac preparations.     

Ca2+ and Mg-ATP activated potassium channels from rat pulmonary artery

The hyperpolarizatlon-activated current (I_f) was recorded from single myocytes dissociated from rabbit sinoatrial node. Although I_f is usually carried by both Na⁺ and K⁺, removal of the minor K⁺ component from physiological saline suppresses inward component. This inward Na⁺ current through I_f channel increases on raising the extracellular K⁺ concentration. The Na⁺ conductance relative to K⁺ conductance (P_Na/P_K), as measured from the reversal potential, increases and saturates near 5 mM K⁺. This effect is different from the current increase caused by raising the concentration of carrier ion K⁺, which saturates at 70 mM with a half-maximal value (K_1/2) of 10 mM. It is suggested that the I_f channel has multiple, interactive binding sites for cation permeation.     

Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma×glioma cells

 By using the whole-cell patch-clamp technique, an inwardly rectifying potassium current, which resembled the ”classic” inward-rectifying potassium current (I _KIR) of other cells in terms of electrophysiological and pharmacological properties, was identified in db-cAMP-differentiated NG108-15 cells. First, the current was dependent on voltage and time. It could be elicited by applying an initial depolarizing prepulse and a subsequent hyperpolarizing command pulse to the cell. The amplitude of the current depended on both the prepulse and the command pulse and increased with the hyperpolarization of the command pulse as well as the depolarization and the prolongation of the prepulse. The activation and inactivation of the current could be fitted well by single-exponential functions and increased with the hyperpolarization of the membrane. Second, the current was dependent on the extracellular potassium concentration ([K⁺]_o). Elevation of [K⁺]_o resulted in a marked increase in the current amplitude and a positive shift of the peak-current/voltage curve as well as the reversal potential. A tenfold increase of [K⁺]_o introduced an ≈43-mV shift of the reversal potential, indicating that the current was carried mainly by K⁺. The conductance (g/g _Max) of the current was also dependent on the [K⁺]_o and increased with increases in [K⁺]_o in a manner approximately proportional to the square-root of [K⁺]_o. Finally, the current was sensitive to Cs⁺ (1 mmol/l), Ba²⁺ (1 mmol/l) and quinidine (0.2 mmol/l); whereas, two typical potassium channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), were weak blockers and reduced the current at high concentration (>10 mmol/l). It was also observed that the current was depressed by Cd²⁺ (1 mmol/l) and Co²⁺ (1 mmol/l) and increased by perfusing the cell with Ca²⁺-free solution. Thus, except for the sensitivity to Cd²⁺, Co²⁺ and Ca²⁺, the current displayed most of the hallmarks described for the ”classic”I _KIR. In conclusion, there appears to be a voltage-dependent I _KIR-type inward rectifier in db-cAMP-differentiated NG108-15 cells. Received: 3 June 1996 / Received after revision: 31 October 1996 / Accepted: 1 November 1996     

Effects of dopamine on ion transport across the rat distal colon

Dopamine (5·10^–6–5·10^–4 M) induced a concentration-dependent decrease in short-circuit current (I_sc) across the rat distal colon. This response was preceded by a transient and inconsistent increase in I_sc. The -adrenoceptor blocker phentolamine and the inhibitors of dopamine-2-like (D₂-like) receptors L-741,626 and L-745,870 inhibited the dopamine response, suggesting a contribution of adrenergic and dopaminergic receptors. The decrease in I_sc evoked by dopamine was inhibited by bumetanide, an inhibitor of the basolateral Na⁺-K⁺-2 Cl^– cotransporter responsible for the uptake of K⁺, and by quinine, a blocker of apical K⁺ channels, indicating that stimulation of K⁺ secretion contributes to the measured change in I_sc. In patch-clamp experiments dopamine hyperpolarized the membrane and increased cellular K⁺ current. This response was not concomitant with a change in the intracellular [Ca²⁺] as demonstrated in parallel fura-2 experiments. These results demonstrate that dopamine, like other catecholamines, stimulates colonic K⁺ secretion.     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

A hyperpolarization-activated cation current (I h) contributes to resting membrane potential in rat superior cervical sympathetic neurones

 Using perforated-patch voltage-clamp recording, a prominent hyperpolarization-activated inward cation current (I _h) has been identified in dissociated, cultured and replated, superior cervical sympathetic (SCG) neurones from 17-day-old rats. I _h was identified as a slowly activated inward current on hyperpolarizing from –60 mV, with an extrapolated null potential (in 3 mM [K⁺]_out) of –42 mV. The activation range for I _h was –40 to –100 mV, with a half-activation voltage (V _0.5) of –63 mV. The current was suppressed by 1 mM Cs⁺ but not by 1 mM Ba²⁺. The reversal potential for the current change induced by Cs⁺ agreed with the null potential for I _h. I _h conferred strong inward rectification to the current-voltage curve negative to –55 mV in both voltage-clamp and current-clamp recording. This inward rectification was reduced by 1 mM Cs⁺. In a sample of eight cells with initial resting membrane potentials between –51 and –64 mV, Cs⁺ increased the resting potential of all cells by between 2.5 and 21 mV. These results indicate that I _h contributes a tonic inward (depolarizing) component to the maintenance of the resting membrane potential in SCG neurones. Received: 16 January 1998 / Received after revision and accepted: 1 April 1998     

Changes in extracellular potassium and calcium in rat cerebellar cortex related to local inhibition of the sodium pump

Extracellular K⁺, Ca²⁺, and Na⁺ ([K⁺]_e, [Ca²⁺]_e, [Na⁺]_e) were recorded with ion selective microelectrodes in the cerebellar cortex of urethane-anesthetized rats. Superfusion of the cerebellum with artificial cerebrospinal fluid containing K-strophanthidin (10^–6–10^–4 mol/l) or other cardioactive steroids, known to be inhibitors of the sodium/potassium pump, had the following effects: elevation of resting [K⁺]₃, reduction of poststimulus K⁺-undershoots, decrease of resting [Ca²⁺]_e and [Na⁺]_e. For instance, at 3×10^–5 mol/l K-strophanthidin within the superfusion solution (the unknown intracerebellar concentration being certainly much smaller), [K⁺]_e was elevated up to 130% and [Ca²⁺]_e reduced to 70% of their resting values. Iontophoretic K⁺-pulses were enhanced in amplitude at the same time. Control experiments with iontophoretic TMA application demonstrated that the glycoside effects were not due (or in higher concentrations only partly due) to shrinkage of the extracellular fluid volume. When tetrodotoxin (10^–7 mol/l) or Mn²⁺ (1–3 mmol/l) were additionally superfused, K-strophanthidin effects were qualitatively similar, though quantitatively smaller. This indicates that part of the effects were indirect via neuronal activity evoked by the blockade of the sodium pump. The experiments show that reduction of sodium pump activity in cerebellar cortex has rapid and serious consequences on the distribution of potassium and calcium in the extracellular space, resulting in an alteration of neuronal circuit excitability.     

Insulin effects on ouabain binding in A6 renal cells

 The effects of insulin on the Na⁺-K⁺-ATPase pump of the basolateral membrane of tight epithelia were evaluated by measuring transepithelial transport and [³H]ouabain binding in cultured A6 kidney cells. [³H]Ouabain binding in epithelia incubated in either K⁺-containing or K⁺-free solutions was measured. Insulin induced increases in transepithelial sodium transport, as measured by the short-circuit current (I _sc), and in the initial rate of [³H]ouabain binding determined when the preparation was bathed in K⁺-containing solutions. However, when initial [³H]ouabain binding in tissues incubated in K⁺-free solutions was measured the stimulation of the initial rate of [³H]ouabain binding caused by insulin was markedly reduced. Incubating the apical side of the epithelium with either amiloride or Na⁺-free solutions also reduced or abolished the increase in the initial rate of [³H]ouabain binding caused by insulin. Equilibrium binding measurements showed that insulin did not increase the maximum number of [³H]ouabain-binding sites in tissues incubated with either normal K⁺ or K⁺-free solutions. These results indicate that the increase in the initial rate of [³H]ouabain binding under transporting conditions is due to an effect on the binding kinetics of ouabain, probably related to an increased rate of Na⁺ entry, rather than to an increase in the number of Na⁺-K⁺-ATPases in the basolateral membrane. Cycloheximide inhibited both the increase in I _sc and the increase in the initial rate of [³H]ouabain binding caused by insulin in epithelia incubated in K⁺-containing solutions. However, cycloheximide was without effect on the initial rate of [³H]ouabain binding in insulin-treated tissues incubated in K⁺-free solution. This finding suggests that the cycloheximide-sensitive step of the action of insulin is related to Na⁺ delivery to the pump. Received: 2 October 1995 / Received after revision: 6 January 1997 / Accepted: 13 January 1997     

Potassium-transporting proteins in skeletal muscle: cellular location and fibre-type differences

Potassium (K⁺) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K⁺ concentration ([K⁺]_e) to values higher than approx. 10 mm significantly reduce force development in unfatigued skeletal muscle. Several in vivo studies have shown that [K⁺]_e increases progressively with increasing work intensity, reaching values higher than 10 mm . This increase in [K⁺]_e is expected to be even higher in the transverse (T)-tubules than the concentration reached in the interstitium. Besides the voltage-sensitive K⁺ (K_v) channels that generate the action potential (AP) it is suggested that the big-conductance Ca²⁺-dependent K⁺ (K_Ca1.1) channel contributes significantly to the K⁺ release into the T-tubules. Also the ATP-dependent K⁺ (K_ATP) channel participates, but is suggested primarily to participate in K⁺ release to the interstitium. Because there is restricted diffusion of K⁺ to the interstitium, K⁺ released to the T-tubules during AP propagation will be removed primarily by reuptake mediated by transport proteins located in the T-tubule membrane. The most important protein that mediates K⁺ reuptake in the T-tubules is the Na⁺,K⁺-ATPase α₂ dimers, but a significant contribution of the strong inward rectifier K⁺ (Kir2.1) channel is also suggested. The Na⁺, K⁺, 2Cl⁻ 1 (NKCC1) cotransporter also participates in K⁺ reuptake but probably mainly from the interstitium. The relative content of the different K⁺-transporting proteins differs in oxidative and glycolytic muscles, and might explain the different [K⁺]_e tolerance observed.     

Electrophysiological properties of membrane currents in single myometrial cells isolated from pregnant rats

The whole-cell voltage-clamp method was applied to single smooth muscle cells prepared from the longitudinal layer of the pregnant rat myometrium (17–20 days of gestation). It was found that the transient inward current mainly consists of Ca²⁺ current, because the removal of Ca²⁺ ions from the external medium and 10 M nifedipine eliminated this inward current. Its steady-state inactivation curve was obtained by the standard method, in which the membrane potential of half inactivation and the slope factor were estimated to be –58.0±4.9 mV (n=11) and 8.9±1.4 mV (n=11), respectively. In a small number of preparations (in 2 out of 30 preparations), there remained a very fast inward current in Ca²⁺-free medium containing Mg²⁺. Tetrodotoxin (TTX, 10 M) can abolish this current, suggesting that the channel for this current is equivalent to the Na⁺ channel in nerve cells. Two major phases of outward currents were identified by voltage jumps from negative holding levels to more positive levels. The first phase was a fast transient outward current. This current remained intact after external tetraethylammonium (TEA, 20 mM) was added. Following the transient current, a large delayed rectified outward current reached its peak over a period of 50 ms and then decayed. The reversal potential for this outward current was determined by observing the change of polarity of the tail currents with the change in extracellular K⁺ concentration ([K⁺]₀). The slope for the change of reversal potential per ten-fold change in [K⁺]₀ is 57.7 mV at more than 23.2 mM [K⁺]_o, indicating that this current is mostly carried by K⁺ ions. Voltage-dependent inactivation of the delayed rectified outward current was determined by the standard method. The membrane potential for half inactivation and the slope factor were estimated to be –42.8±3.9 mV (n=3) and 10.1±1.5 mV (n=3), respectively. External TEA (20 mM) effectively eliminated the delayed rectified outward currents. Nifedipine (10 M) suppressed not only Ca²⁺ current but also outward K⁺ currents.