Maurotoxin: a potent inhibitor of intermediate conductance Ca2+-activated potassium channels.

Maurotoxin, a 34-amino acid toxin from Scorpio maurus scorpion venom, was examined for its ability to inhibit cloned human SK (SK1, SK2, and SK3), IK1, and Slo1 calcium-activated potassium (K(Ca)) channels. Maurotoxin was found to produce a potent inhibition of Ca(2+)-activated (86)Rb efflux (IC(50), 1.4 nM) and inwardly rectifying potassium currents (IC(50), 1 nM) in CHO cells stably expressing IK1. In contrast, maurotoxin produced no inhibition of SK1, SK2, and SK3 small-conductance or Slo1 large-conductance K(Ca) channels at up to 1 microM in physiologically relevant ionic strength buffers. Maurotoxin did inhibit (86)Rb efflux (IC(50), 45 nM) through, and (125)I-apamin binding (K(i), 10 nM) to SK channels in low ionic strength buffers (i.e., 18 mM sodium, 250 mM sucrose), which is consistent with previous reports of inhibition of apamin binding to brain synaptosomes. Under similar low ionic strength conditions, the potency for maurotoxin inhibition of IK1 increased by approximately 100-fold (IC(50), 14 pM). In agreement with its ability to inhibit recombinant IK1 potassium channels, maurotoxin was found to potently inhibit the Gardos channel in human red blood cells and to inhibit the K(Ca) in activated human T lymphocytes without affecting the voltage-gated potassium current encoded by Kv1.3. Maurotoxin also did not inhibit Kv1.1 potassium channels but potently blocked Kv1.2 (IC(50), 0.1 nM). Mutation analysis indicates that similar amino acid residues contribute to the blocking activity of both IK1 and Kv1.2. The results from this study show that maurotoxin is a potent inhibitor of the IK1 subclass of K(Ca) potassium channels and may serve as a useful tool for further defining the physiological role of this channel subtype.

Propafenone inhibition of human atrial myocyte repolarizing currents.

This study was aimed at defining cellular electropharmacologic effects of propafenone on repolarizing currents in human atrial myocytes. Whole-cell patch-clamp of enzymatically isolated atrial myocytes from 11 cardiac surgical patients aged between 29 days and 74 years revealed potent time- and concentration-dependent (IC50 = 4.8 +/- 0.4 mumol/l), but age-, voltage-, and frequency-independent propafenone inhibition of transient outward current. Time course of apparent transient outward current inactivation was best described by a single exponential process in the absence of propafenone and by a double exponential model in its presence, with drug-concentration-dependent acceleration of the fast exponential component. Neither voltage dependence of steady-state transient outward current inactivation nor time course of recovery from inactivation was affected by propafenone. Significant inhibition (P < 0.05) of the ultra-rapidly activating delayed rectifier and inwardly rectifying currents was observed only in the presence of > or = 10 mumol/l propafenone. These actions of propafenone could explain its repolarization prolonging effect and might contribute to clinical electrophysiologic responses which have been documented in patients of all ages.

Characterisation of transient outward current in young human atrial myocytes.

OBJECTIVE: The aim was to characterise the development of the transient outward current (Ito) in atrial myocytes of infants and children. METHODS: Whole cell voltage clamp was used to study outward currents in enzymatically isolated atrial myocytes from infants and children ranging in age from 3 days to 13.2 years. RESULTS: A transient inactivating current characteristic of Ito was observed in 71 myocytes from 22 patients aged 3 days to 13.2 years, including a 10 day old infant born prematurely at 33 weeks gestation. There was no discernible developmental trend in Ito current density [10.74(SEM 0.65) pA.pF-1 at +40 mV, n = 71 cells from 22 patients] or voltage dependence of inactivation, newborn values being similar to those in older children, and in adults reported elsewhere. A developmental reduction in total outward current density was attributable entirely to diminution of the non-inactivating steady state current component. The Ito time course of inactivation showed an apparent maturational evolution, with the youngest infants having slightly but significantly slower inactivation kinetics. The kinetics of Ito recovery from inactivation were well described by a single exponential model with no appreciable developmental trend in time course. CONCLUSIONS: Ito is expressed in human atrial myocytes from early infancy and does not show significant developmental changes in current density. The relative contribution of Ito to myocyte repolarisation might increase with age as a result of diminution in the non-inactivating current component. There is an apparent slight maturational acceleration in the time course of Ito inactivation but not in recovery from inactivation, perhaps excluding the latter as a mechanism for the previously reported functional unavailability of Ito in young human atrial muscle.

4-Aminopyridine binding and slow inactivation are mutually exclusive in rat Kv1.1 and Shaker potassium channels.

In the present study we have used two-electrode voltage-clamping of Xenopus oocytes expressing either Kv1.1 or Shaker B (ShB) delta 6-46 K+ channels to examine the effects of 4-aminopyridine (4-AP) on the process of slow inactivation. Neither of these channels exhibits fast inactivation. Channel activation was required for block by 4-AP in both channel types. In the absence of drug, inactivation of Kv1.1 and ShB delta 6-46 channels at 0 mV was biexponential [tau fast = 7.8 +/- 0.3 sec, tau slow = 33.9 +/- 0.9 sec, and Aslow/(Afast + Aslow) = 0.79 +/- 0.02 (n = 10) for Kv1.1 and tau fast = 3.5 +/- 0.4 sec, tau slow = 13.1 +/- 1.8 sec, and Aslow/(Afast + Aslow) = 0.35 +/- 0.06 (n = 3) for ShB delta 6-46]. In the presence of 4-AP, the rates of inactivation of Kv1.1 and ShB delta 6-46 were markedly slowed, resulting in a crossover phenomenon where, in the presence of drug, the outward current was smaller than control at the beginning of the depolarizing pulse but crossed over during the pulse to become larger than the control. The most obvious change induced by 0.2 mM 4-AP was a 2-fold slowing of the slow phase of inactivation [tau fast = 3.9 +/- 1.1 sec, tau slow = 67.1 +/- 3.6 sec, and Aslow/(Afast + Aslow) = 0.85 +/- 0.04 (n = 4) for Kv1.1 and tau fast = 3.5 +/- 0.4 sec, tau slow = 23.7 +/- 2.6 sec, and Aslow/(Afast + Aslow) = 0.75 +/- 0.02 (n = 3) for ShB delta 6-46, in the presence of 0.2 mM 4-AP]. In addition, there was a significant increase in the contribution of the slower phase of inactivation of ShB delta 6-46 channels in the presence of 4-AP. The slowed inactivation in the presence of 4-AP was accompanied by removal of 4-AP block. These results are consistent with the processes of 4-AP block and slow inactivation of Kv1.1 and ShB delta 6-46 channels being mutually exclusive.

Aminopyridine block of Kv1.1 potassium channels expressed in mammalian cells and Xenopus oocytes.

The mechanism by which aminopyridines (APs) block cloned Kv1.1 K+ channels expressed either in the mammalian Sol-8 muscle cell line or in Xenopus oocytes was investigated using whole-cell patch-clamp and two-electrode voltage-clamp techniques. When Sol-8 cells were exposed to 4-AP (30 microM to 1 mM) or 3-AP (300 microM to 10 mM) for 1-2 min at a holding potential of -80 mV, delayed rectifier K+ currents activated by the first depolarization to +40 mV showed a small reduction in peak amplitude but exhibited a rapid decay phase that was absent in control records. However, currents elicited by subsequent pulses showed maximal block throughout the pulse. These results suggest that 4-AP requires the channel to be activated before block can occur readily. After a 10-min washout of 4-AP in the absence of channel activation, the current elicited at the beginning of the first depolarizing pulse was similar to that observed during maximal block. However, during the pulse the current increased in an exponential manner, to an amplitude similar to that seen in the absence of 4-AP. Subsequent pulses elicited currents with profiles similar to that observed in controls. These results suggest that channel activation is also required for unblock (i.e., 4-AP is trapped when the channel closes). Block of Kv1.1 channels by 4-AP was voltage dependent. The magnitude of block decreased progressively as the membrane potential was depolarized to voltages more positive than -20 mV. The concentration producing half-maximal inhibition (IC50) of Kv1.1 currents in Sol-8 cells at +40 mV at physiological pH (7.2) was 89 microM for 4-AP (pKa = 9.2) and 2.2 mM for 3-AP (pKa = 6.0). However, when the intracellular pH was lowered to 6.0 the IC50 for 3-AP decreased to 290 microM. These results are consistent with the charged form of the APs being the active species.

K+ channel blocking actions of flecainide compared with those of propafenone and quinidine in adult rat ventricular myocytes.

The K+ channel blocking action of the class Ic antiarrhythmic agent flecainide was compared with that of propafenone and quinidine in isolated adult rat ventricular myocytes by using the whole-cell patch-clamp technique. In rat ventricular myocytes, depolarization activates both an inactivating (ITO) and a maintained (IK) outward K+ current. Flecainide, propafenone and quinidine all were potent inhibitors of ITO with IC50s of 3.7, 3.3 and 3.9 microM, respectively. Flecainide and quinidine were less potent inhibitors of IK than was propafenone with IC50s of 15 and 14 microM compared with an IC50 of 5 microM for propafenone. By contrast with their effects on outward currents, these agents produced little or no inhibition of the inward rectifier K+ current, even when present at 300 microM. All three agents produced a concentration-dependent increase in the rate of inactivation of ITO but they only produced minor hyperpolarizing shifts (approximately 3 mV) in the voltage dependence of steady-state inactivation. Although propafenone had little effect on the rate of ITO recovery from inactivation (tau CONTROL = 64 +/- 5 ms; tau PROPAFENONE = 84 +/- 9 ms), ITO recovery in the presence of flecainide and quinidine was biexponential; it exhibited an additional slow component (tau FAST = 67 +/- 5 ms and tau SLOW = 2580 +/- 1500 ms for flecainide; tau FAST = 55 +/- 5 ms and tau SLOW = 871 +/- 99 ms for quinidine). Consistent with these observations, flecainide and quinidine, but not propafenone, produced use-dependent block of ITO at a stimulation frequency of 1 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of 4-aminopyridine block of the transient outward K+ current in adult rat ventricular myocytes.

The blocking action of 4-aminopyridine (4-AP) on the transient outward K+ current (ITO) in isolated rat ventricular myocytes was studied using the whole-cell configuration of the patch-clamp technique. 4-AP inhibition of ITO was concentration-dependent with half-maximal block occurring at 0.2 mM. At high concentrations (> 1 mM), 4-AP appeared to slow both the activation and inactivation phases of ITO. This resulted in a crossover phenomenon where in the presence of 4-AP the outward current was less than control at the beginning of a depolarizing pulse but crossed over during the pulse to become greater than control. Inhibition of ITO by 4-AP was voltage-dependent. Steady-state block of ITO by 4-AP was greatest at or near resting membrane potentials (i.e., -70 mV) but decreased with membrane depolarization. The voltage-dependence of block was steep and was well described by a Boltzmann relationship with a slope factor of approximately 4 mV. The midpoint potential for block was dependent on the concentration of 4-AP, being -41.6 +/- 0.4 mV (n = 9), -40.7 +/- 1.3 mV (n = 6), -34.0 +/- 1.6 mV (n = 5) and -30.1 +/- 0.2 (n = 15) at 0.3, 1, 3 and 10 mM, respectively. The midpoint potential for activation was -12.6 mV and was -46.9 mV for inactivation. The concentration-dependence of the voltage-dependence of 4-AP block can be explained by assuming that the sequential closed states through which the channel passes during activation exhibit successively lower affinities for 4-AP. Onset of ITO block by 4-AP was slow. The association (kON) and dissociation. (kOFF) rate constants for binding at -70 mV were: kON = 207 M-1 s-1 and kOFF = 0.090 s-1. The time constant for unblock (tau UNBLOCK) of ITO at 0 mV was independent of 4-AP concentration indicating that there was no binding of 4-AP at this potential. kOFF (1/tau UNBLOCK) at 0 mV was 2.4 s-1 which is approximately 25-fold faster then at -70 mV. The results suggest that 4-AP binds most strongly to closed channels with the inactivation gate open. The conformational changes that occur during channel opening induce a decrease in affinity for 4-AP so that when the channel is in the open state, 4-AP binding is at its weakest. The processes of 4-AP block and inactivation appear to be mutually exclusive.

Dequalinium: a potent inhibitor of apamin-sensitive K+ channels in hepatocytes and of nicotinic responses in skeletal muscle.

The bisquaternary compound dequalinium has been tested for its ability to inhibit the loss of K+ which angiotensin II causes in guinea-pig hepatocytes and which occurs through apamin-sensitive Ca(2+)-activated K+ (SKCa) channels. Dequalinium blocked angiotensin II-evoked K+ loss with an IC50 of 1.5 +/- 0.1 microM and also inhibited 125I-monoiodoapamin binding with a KI of 1.1 +/- 0.1 microM. It is the most active non-peptide SKCa blocker so far described. The neuromuscular blocking agent vecuronium was also tested, and proved to be considerably less effective (IC50, 4.5 +/- 0.3 microM; KI, 3.6 +/- 0.5 microM). Dequalinium was also examined for its actions at nicotinic receptors in skeletal muscle and was found to be a potent, non-competitive antagonist of carbachol contractions of the frog rectus abdominis. In the frog cutaneous pectoris muscle, end-plate depolarizations induced by carbachol became smaller and more transient in the presence of dequalinium at 10 nM. However, contractions of the frog sartorius and rat diaphragm in response to nerve stimulation were inhibited only by concentrations > 1 microM. These apparently discrepant effects of dequalinium on nicotinic responses could be explained either by open channel block of slow onset or by 'stabilization' of the desensitized state of the receptor. The potency of dequalinium will make it a useful agent for the study of nicotinic receptors as well as of SKCa channels.

Differential inhibition of potassium currents in rat ventricular myocytes by capsaicin.

OBJECTIVE: Capsaicin is a pungent irritant present in peppers of the Capsicum family. Its major target of action is believed to be sensory neurones. Capsaicin has also been shown to prolong cardiac action potential in atrial muscle, perhaps by local release of calcitonin gene related peptide which in turn enhances inward calcium currents. However, capsaicin has been shown to inhibit K+ current in neurones. Since such an action could contribute to action potential prolonging activity of capsaicin in heart, the aim of the study was to examine the effects of capsaicin on cardiac K+ currents. METHODS: Ionic currents and action potentials were examined in isolated adult rat ventricular myocytes using the whole cell variant of the patch clamp technique at 25 degrees C. RESULTS: Capsaicin (10 microM) increased the action potential duration (APD50) from 45 ms to 166 ms. This effect was associated with an inhibition of three distinct K+ currents. The decreasing rank order of potency was: transient outward K+ current (ITO, IC50 = 6.4 microM), a voltage dependent non-inactivating outward current (IK, IC = 11.5 microM), and the inward rectifier K+ current (IK1, IC50 = 46.9 microM). Capsaicin induced block of ITO was characterised by a decrease in the peak current amplitude and an increase in the rate of inactivation. The inactivation of ITO in the absence of capsaicin was well described by a single exponential [tau = 77 (SEM 2) ms at +40 mV, n = 10]. However, in the presence of 10 microM capsaicin inactivation was best described by the sum of two exponentials [tau FAST = 4.4(0.5) ms; tau SLOW = 92.4(3.0) ms, n = 10] with the fast component contributing 46(2)% of the total decay. A small but consistent hyperpolarising shift (approximately 3 mV) in the steady state voltage dependence of inactivation of ITO was induced by 10 microM capsaicin. Capsaicin had no effect on the rate of ITO recovery from inactivation (tau = 49 ms and 48 ms for control and drug respectively). The capsaicin analogue, resiniferatoxin, which as an irritant is up to 10(4)-fold more potent than capsaicin, had no effect on any of the K+ currents when present at concentrations of up to 10 microM. In contrast another capsaicin analogue, zingerone (30 microM) blocked ITO by 52(12)% and IK by 35%. CONCLUSIONS: Capsaicin produces a prolongation of the rat ventricular action potential, an effect which is associated with inhibition of potassium currents.

Identification of RBK1 potassium channels in C6 astrocytoma cells.

Ionic currents in C6 astrocytoma cells were studied using the patch clamp technique under the whole cell configuration. A delayed rectifier K+ current with an amplitude of approximately 1 nA at +50 mV was observed in 86% (92/107) of the cells examined. This K+ current resembled the delayed rectifier present in type-1 and type-2 astrocytes in vitro and could be inhibited by a variety of K+ channel blockers, including TEA (IC50:0.5 mM), 4-aminopyridine (IC50:0.2 mM), MCD peptide (IC50:52 nM), dendrotoxin I (IC50:9 nM), and charybdotoxin (74% inhibition at 50 nM). Northern blot analysis, cloning of cDNA and subsequent sequencing showed that the C6 cell delayed rectifier K+ channel is equivalent to the RBK1 K+ channel derived from a rat brain cDNA library. The level of RBK1 transcripts in C6 cells was comparable to that reported in rat brain. The C6 delayed rectifier K+ channel is probably a homomeric RBK1 K+ channel judging from its pharmacological properties which are similar to the RBK1 channel expressed in Xenopus oocytes. Some C6 cells also expressed a transiently activated outward K+ current (IA). This current was found in less than 50% of the cells and in general contributed no more than 8% of the total outward current. No voltage-dependent inward Na+ or Ca2+ currents or inwardly rectifying K+ currents were observed in over 100 C6 cells examined. The present results show that the dominant voltage gated ionic current in C6 cells is the RBK1 delayed rectifier K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective inhibition of potassium currents in rat ventricle by clofilium and its tertiary homolog.

The effects of clofilium and its tertiary homolog, LY97119 (LY), on K+ currents in isolated ventricular myocytes from adult rat were examined using the whole-cell patch-clamp technique. Acute exposure (less than 30 min) to high concentrations of clofilium (30-100 microM) produced a slowly developing reduction in the peak of the transient outward current (Ito) and an apparent increase in the rate of current inactivation. In addition, inhibition exhibited a marked dependence on the frequency of stimulation (i.e., use-dependent inhibition). For "short" exposure times (i.e., less than 30 min), steady-state inhibition was not attained. Therefore, myocytes were preincubated with clofilium for at least 3 hr before use. Under these conditions, the median inhibitory concentration for steady-state use-dependent inhibition of Ito was 0.5 microM. Recovery of Ito from use-dependent inhibition followed a biexponential time course; tau fast = 75 msec, tau slow = 17 sec. The relative magnitude of the slow component (but not its time constant) increased with higher clofilium concentrations. LY, like clofilium, also induced a time-dependent inhibition of Ito (median inhibiting concentration 0.9 microM). However, unlike clofilium, steady state with LY was reached within 5 min. Furthermore, LY (3 microM) produced very little use-dependent inhibition (29% at 1 Hz compared with 92% for clofilium). This is possibly due to a "fast" unbinding rate upon repolarization (tau = 1.8 s). In contrast to clofilium, LY (1-10 microM) also inhibited the inward rectifier (IK1). The present results suggest that inhibition of Ito may contribute to clofilium's class III antiarrhythmic action.

Block of single batrachotoxin-activated Na+ channels by clofilium.

The effects of clofilium on single batrachotoxin-activated Na+ channels from rabbit skeletal muscle, incorporated into planar bilayers, were studied under symmetrical 200 mM NaCl conditions. Internally applied clofilium (0.3-30 microM) induced long lasting closures, with a mean duration of approximately 450 msec at +50 mV. The fraction of time spent in the clofilium-induced closed state was concentration dependent, with an equilibrium dissociation constant (Kd) of 3.4 microM at +50 mV. Kinetic analysis showed that both open and closed time distributions were well described by single exponentials, with respective time constants of tau o and tau C. As expected for an open channel blocker, 1/tau o increased linearly with increasing clofilium concentration, whereas 1/tau c remained relatively constant. Inhibition of batrachotoxin-activated Na+ channels by clofilium exhibited a strong voltage dependence. The binding affinity of clofilium increased about 10-fold upon depolarization from -50 mV to 50 mV. Competition studies using quaternary and tertiary local anesthetics showed that clofilium and local anesthetics probably share a common receptor site located about halfway across the membrane electrical field. Together, our results demonstrate that clofilium is a potent Na+ channel blocker in bilayers and its action is similar to that of other local anesthetics characterized previously.

Bupivacaine inhibits the transient outward K+ current but not the inward rectifier in rat ventricular myocytes.

The effects of bupivacaine on K+ currents in isolated rat ventricular myocytes were examined using the whole-cell patch-clamp technique. Bupivacaine at concentrations greater than 3 microM produced both a reduction in the peak current amplitude and a marked increase in the rate of inactivation of the transient outward current (Ito). Examination of its time course showed that there was no inhibition before the beginning of a depolarizing pulse. However, upon continuous depolarization (i.e., during channel opening) inhibition of Ito developed in an exponential manner, the rate and magnitude of which were dependent on bupivacaine concentration. The IC50 for inhibition of Ito was 22 microM. Bupivacaine had no effect on the voltage-dependence of steady-state inactivation or the rate of recovery from inactivation. The (+)- and (-)-stereoisomers of bupivacaine were equipotent indicating that there is no stereoselectivity to the inhibition of Ito. Increasing the hydrophobicity of the tertiary amine on bupivacaine greatly enhanced its potency. Thus, octylacaine (1-octyl-2',6'-pipecoloxylidide) (C8-N) was 6 times more potent than bupivacaine (C4-N) and 200 times more potent than mepivacaine (C1-N). In contrast to their effects on Ito, bupivacaine (1 mM) and octylacaine (100 microM) failed to produce any block of the inward rectifier K+ current. However, mepivacaine (3 mM) reduced inward rectifier K+ current reversibly by approximately 50%. These results suggest that inhibition of Ito may contribute to bupivacaine-induced cardiotoxicity.

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier.

The effect of forskolin on voltage-activated Na+ and K+ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltage-clamp technique. Peak Na+ currents (INa) were reduced by 35% and the rate of decline of Na+ current during continuous depolarization was accelerated following treatment with 450 microM forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1-10 Hz, a further inhibition of INa (approximately 60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV (tau approximately 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na+ channel in the blocking process. Inhibition of the delayed K+ current (IK) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC50 approximately 80 microM) and a concentration-independent acceleration of current inactivation. A similar inhibition of IK was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition IK and perhaps INa follows directly from drug binding and is not a consequence of AC activation.

Toxins in the characterization of potassium channels.

Several recently characterized toxins (apamin, charybdotoxin, dendrotoxin and noxiustoxin) are proving invaluable for establishing what kinds of potassium channel are expressed in neurones, and what the roles of the channels might be.

Palytoxin induces a relatively non-selective cation permeability in frog sciatic nerve which can be inhibited by cardiac glycosides.

The effect of palytoxin (PTX) on compound resting potential and compound action potential of frog sciatic nerve was studied using the sucrose-gap technique. PTX irreversibly depolarized the compound resting potential and reduced the amplitude of the compound action potential. PTX evoked a marked depolarization when extracellular Na+ was replaced by Li+, Cs+ and the organic cations methylammonium, hydroxylammonium, and methylhydroxylammonium but not by tetramethylammonium, tetraethylammonium, choline or the divalent cations, Ca2+ and Ba2+. The maintained depolarization was not sensitive to inhibition by saxitoxin (300 nM) or procaine (10 mM). The depolarization was inhibited by ouabain or cymarin but not by the aglycon, strophanthidin. However, strophanthidin did antagonize the inhibitory action of cymarin which suggests that PTX and cardiac glycosides do not share an identical binding site but there may be some overlap. We conclude that in frog sciatic nerve, PTX interacts with the (Na+-K+) pump to induce the opening or formation of a relatively non-selective cation pore within or near the pump protein.

Effect of channel blockers on potassium efflux from metabolically exhausted frog skeletal muscle.

1. 86Rb and 42K have been used to assess potassium exchange in frog skeletal muscle which had been metabolically exhausted by electrical stimulation (1 Hz) after treatment with 2 mM-cyanide and 1 mM-iodoacetate. These conditions led to the development of rigor. 2. Poisoning by itself induced a small but variable increase in tracer efflux. Complete mechanical exhaustion subsequent to electrical stimulation was, however, accompanied by a 5-6 fold increase in the rate coefficient for both 86Rb and 42K efflux. In the case of rubidium this was maintained for at least 20 min and often for up to 1 h. 3. The increase in tracer efflux induced by metabolic exhaustion was inhibited by barium (0.03-5 mM) in a reversible and concentration-dependent manner. Inhibition was also observed with glibenclamide (3-100 microM), tolbutamide (0.3-2 mM), TEA (5-100 mM) and the local anaesthetics lignocaine (1-3 mM) and tetracaine (1 mM). Quinine produced a dual response consisting of an inhibitory component which was most clearly seen at low concentrations (0.3 mM) and an enhancement of tracer efflux that became increasingly dominant at higher concentrations (1-10 mM). 4. Both apamin (30 and 100 nM) and Israeli scorpion (Leiurus quinquestriatus) venom (16 micrograms ml-1) produced little or no block of the tracer efflux activated by metabolic exhaustion. Similarly 4-aminopyridine (3 mM) and decamethonium (0.3 mM) were without obvious effect. 5. It is concluded that metabolic exhaustion of frog skeletal muscle leads to an increased permeability to both 42K and 86Rb. Our results with channel blockers suggest that this K+ permeability can be attributed neither to the delayed rectifier nor to an apamin- or charybdotoxin-sensitive calcium-activated K+ permeability (PK(Ca) but may be predominantly due to activation of ATP-sensitive channels similar to those found in the beta-cells of pancreatic islets.

Identification of two toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channel.

Two polypeptide toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channels have been identified and partially purified. One toxin, at 50-100 ng/ml, blocks apamin-sensitive potassium fluxes in hepatocytes and inhibits [125I]monoiodoapamin binding. The other, more basic, toxin blocks apamin-insensitive potassium fluxes in erythrocytes at 200 ng/ml and, to our knowledge, is the first toxin shown to block the erythrocyte calcium-activated potassium channel with high affinity. The possible co-identity of this latter toxin with charybdotoxin is discussed.