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Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which a significant fraction of the presynaptic P/Q-type Ca(2+) channels critical to the triggering of neurotransmitter release at the neuromuscular junction (NMJ) are thought to be removed. There is no cure for LEMS, and the current most commonly used symptomatic treatment option is a potassium channel blocker [3,4-diaminopyridine (3,4-DAP)] that does not completely reverse symptoms and can have dose-limiting side-effects. We previously reported the development of a novel Ca(2+) channel agonist, GV-58, as a possible alternative treatment strategy for LEMS. In this study, we tested the hypothesis that the combination of GV-58 and 3,4-DAP will elicit a supra-additive increase in neurotransmitter release at LEMS model NMJs. First, we tested GV-58 in a cell survival assay to assess potential effects on cyclin-dependent kinases (Cdks) and showed that GV-58 did not affect cell survival at the relevant concentrations for Ca(2+) channel effects. Then, we examined the voltage dependence of GV-58 effects on Ca(2+) channels using patch clamp techniques; this showed the effects of GV-58 to be dependent upon Ca(2+) channel opening. Based on this mechanism, we predicted an interaction between 3,4-DAP and GV-58. We tested this hypothesis using a mouse passive transfer model of LEMS. Using intracellular electrophysiological ex vivo recordings, we demonstrated that a combined application of 3,4-DAP plus GV-58 had a supra-additive effect that completely reversed the deficit in neurotransmitter release magnitude at LEMS model NMJs. This reversal contrasts with the less significant improvement observed with either compound alone. Our data indicate that a combination of 3,4-DAP and GV-58 represents a promising treatment option for LEMS and potentially for other disorders of the NMJ.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which a significant fraction of the presynaptic P/Q-type Ca(2+) channels critical to the triggering of neurotransmitter release at the neuromuscular junction (NMJ) are thought to be removed. There is no cure for LEMS, and the current most commonly used symptomatic treatment option is a potassium channel blocker [3,4-diaminopyridine (3,4-DAP)] that does not completely reverse symptoms and can have dose-limiting side-effects. We previously reported the development of a novel Ca(2+) channel agonist, GV-58, as a possible alternative treatment strategy for LEMS. In this study, we tested the hypothesis that the combination of GV-58 and 3,4-DAP will elicit a supra-additive increase in neurotransmitter release at LEMS model NMJs. First, we tested GV-58 in a cell survival assay to assess potential effects on cyclin-dependent kinases (Cdks) and showed that GV-58 did not affect cell survival at the relevant concentrations for Ca(2+) channel effects. Then, we examined the voltage dependence of GV-58 effects on Ca(2+) channels using patch clamp techniques; this showed the effects of GV-58 to be dependent upon Ca(2+) channel opening. Based on this mechanism, we predicted an interaction between 3,4-DAP and GV-58. We tested this hypothesis using a mouse passive transfer model of LEMS. Using intracellular electrophysiological ex vivo recordings, we demonstrated that a combined application of 3,4-DAP plus GV-58 had a supra-additive effect that completely reversed the deficit in neurotransmitter release magnitude at LEMS model NMJs. This reversal contrasts with the less significant improvement observed with either compound alone. Our data indicate that a combination of 3,4-DAP and GV-58 represents a promising treatment option for LEMS and potentially for other disorders of the NMJ.

1. In this study we investigated the expression and function of the K(V)alpha1 subfamily of voltage-gated K(+) channels in terminal arterioles from rabbit cerebral circulation. 2. K(+) current was measured from smooth muscle cells within intact freshly isolated arteriolar fragments. Current activated on depolarisation positive of about -45 mV and a large fraction of this current was blocked by 3,4-diaminopyridine (3,4-DAP) or 4-aminopyridine (4-AP), inhibitors of K(V) channels. Expression of cRNA encoding K(V)1.6 in Xenopus oocytes also generated a 4-AP-sensitive K(+) current with a threshold for activation near -45 mV. 3. Immunofluorescence labelling revealed K(V)1.2 to be specifically localised to endothelial cells, and K(V)1.5 and K(V)1.6 to plasma membranes of smooth muscle cells. 4. K(V) channel current in arteriolar fragments was blocked by correolide (which is specific for the K(V)alpha1 family of K(V) channels) but was resistant to recombinant agitoxin-2 (rAgTX2; which inhibits K(V)1.6 but not K(V)1.5). Heterologously expressed K(V)2.1 was resistant to correolide, and K(V)1.6 was blocked by rAgTX2. 5. Arterioles that were mildly preconstricted and depolarised by 0.1-0.3 nM endothelin-1 constricted further in response to 3,4-DAP, 4-AP or correolide, but not to rAgTX2. 6. We suggest that K(V)alpha1 channels are expressed in smooth muscle cells of terminal arterioles, underlie a major part of the voltage-dependent K(+) current, and have a physiological function to oppose vasoconstriction. K(V)alpha1 complexes without K(V)1.5 appear to be uncommon.

1. The types of K+ channel which determine the membrane potential of arcuate artery smooth muscle cells were investigated by patch-clamp recording from isolated cells and lumenal diameter measurements from intact pressurized renal arcuate arteries. 2. Single cells had a mean resting potential of -38 mV and were depolarized by 130 mM K+ but not by the Cl- channel blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). 3. Iberiotoxin did not affect the resting potential but inhibited spontaneous transient hyperpolarizations. Iberiotoxin or 1 mM tetraethylammonium (TEA+) constricted intact arteries. 3,4-Diaminopyridine (3,4-DAP)-sensitive delayed rectifier K+ (KV) channel current was elicited by depolarization but 3,4-DAP did not affect the resting potential or induce constriction in the intact artery. 4. A voltage-independent K+ current was inhibited by >= 0.1 mM barium (Ba2+) and unaffected by iberiotoxin, glibenclamide, apamin, 3,4-DAP and ouabain. In six out of ten cells, 1 mM Ba2+ depolarized the resting potential, while in the other cells the potential was resistant to all of the K+ channel blockers and ouabain. Ba2+ (0.1-1 mM) constricted the intact artery, but 10 microM Ba2+, 1 microM glibenclamide or 100 nM apamin had no effect. 5. The data suggest that resting potential is determined by background K+ channels, one type being Ba2+ sensitive and voltage independent, and another type being poorly defined due to its resistance to any inhibitor. Large conductance Ca2+-activated K+ (BKCa) and KV channels do not determine the resting potential but have separate functions to underlie transient Ca2+-induced hyperpolarizations and to protect against depolarization past about -30 mV.

1. Changes in the electrical properties of frog motor nerve endings caused by the invasion of an action potential were studied by the perineural recording technique. Two equal supramaximal stimuli separated by a variable time interval were applied to the nerve trunk. The latency and amplitude of the deflections associated with the nodal Na+ current and presynaptic K+ current elicited by the second pulse were compared with control currents. 2. The deflection associated with the presynaptic K+ current elicited in response to the second stimulus was absent at the shortest interstimulus interval and showed a progressive increase in its amplitude as the interstimulus interval was lengthened, reaching values greater than control in most terminals. During the same period the nodal Na+ current did not change. 3. The experimental results were compared with a computer model of the distal axonal segment and its terminal. Response of the model to twin-pulse stimulation was in marked disagreement with the experimental results unless an inactivating K+ channel, with properties derived ad hoc, was incorporated into the simulation. 4. These results suggest that in the first 6-7 ms after a nerve impulse has invaded a frog motor nerve ending, maximal K+ conductance remains below the value at rest due to the fast inactivation of a K+ conductance. Following this, there is a period in which K+ conductance is greater than control values although the basis for this is unknown.

1. The three-microelectrode voltage clamp technique and pharmacological agents were used to examine the properties and functions of potassium currents in squid giant presynaptic terminals. 2. Outward currents consisted of two components: a slow component which activated over hundreds of milliseconds and was blocked by extracellular application of tetraethylammonium (TEA) ions and a more rapidly activating component which was relatively insensitive to extracellular TEA. 3. The more rapid component was studied in isolation by treating presynaptic terminals with extracellular TEA, as well as tetrodotoxin (to block sodium channel currents) and manganese (to block calcium channel currents). The magnitude of this current component was 1-2 mA cm-2 at 0 mV. Rates of activation and deactivation were voltage dependent and little evidence of inactivation was seen for depolarizations less than several seconds in duration. 4. The reversal potential of the current was -70 to -80 mV in normal saline and became more positive with elevated extracellular potassium concentrations, suggesting that potassium is the primary permeant ion. Accumulation of extracellular potassium appeared to be marked during depolarizations that produced significant activation of the current. 5. Extracellular application of 3,4-diaminopyridine (DAP) blocked the current with an apparent dissociation constant of 7 microM at 0 mV. Intracellular applications of DAP and TEA also were effective in reducing this current. These treatments, but not extracellular TEA application, broadened presynaptic action potentials and increased the magnitude and time-to-peak of postsynaptic currents elicited by the broadened presynaptic action potentials. Postsynaptic currents were a sensitive and linear function of action potential duration; a 30% increase in action potential duration increased postsynaptic current amplitude by 190%. 6. Estimation of the magnitude and time course of the presynaptic calcium current, based on previous measurements of calcium channel gating, indicated that action potential broadening produces a large increase in calcium current magnitude. These calculations predict that a 30% increase in presynaptic action potential duration will increase the peak amplitude of the calcium current by approximately 170% and the total amount of calcium entry by approximately 230%. This implies a linear relationship between transmitter release and calcium entry during an action potential and can be explained by assuming that calcium co-operatively triggers release within intracellular domains that do not overlap.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Non-linear membrane ionic current was studied in highly stretched cut frog twitch fibres in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 0.1 mM EGTA and the external solution containing Cl- as the major anion. After the Na+ currents was abolished by TTX in the external solution and the K+ currents were suppressed by external TEA+ and Rb+ and internal Cs+, a delayed outward ionic current with a time course similar to that of the delayed rectifier current was observed during depolarization. 2. The delayed outward ionic current was resistant to 1 mM 3,4-diaminopyridine (3,4-DAP) in the external solution and was unaltered when a fraction of the internal Cs+ was replaced by K+ or Na+, suggesting that the current was not carried by cations flowing through the delayed rectifiers. 3. The delayed outward ionic current was greatly reduced by replacing the external Cl- with CH3SO3-,SO4(2-), glutamate or gluconate, indicating strongly that the current was carried by Cl- flowing through anion channels. The current was also suppressed by 1 mM external 9-anthracenecarboxylic acid (9-ACA). 4. The delayed outward ionic current was reduced by blockers of calcium-dependent Cl- channels, such as SITS and frusemide (furosemide), in a dose- and voltage-dependent manner and by increasing intracellular [EGTA] to 20 mM, suggesting that part of the Cl- current in the muscle fibres could be calcium dependent. 5. The total Cl- current could be dissected into calcium-dependent and calcium-independent components. Each component accounted for roughly half of the total Cl- current. The maximum slope conductance of the calcium-dependent Cl- channels was 60.9 +/- 6.0 microS microF-1 (mean +/- S.E.M., n = 4).

1. The effects of some neutral clinical and experimental general anaesthetics on the resting potential of normal squid axons and squid axons exposed to tetrodotoxin and 3,4-diaminopyridine have been studied. 2. Depolarizations of 1-4 mV were produced by all the anaesthetics at 'clinical' concentrations in the normal axon. Larger depolarizations (5-11 mV) were produced by the same anaesthetic concentrations in axons exposed to tetrodotoxin and 3,4-diaminopyridine. 3. The conductance of axons exposed to tetrodotoxin and either tetraethyl-ammonium or 3,4-diaminopyridine in zero Na+, 430 mM-K+ artificial sea water was examined by voltage clamp and AC bridge techniques. 4. The evidence that this conductance is due predominantly to K+ is discussed. 5. Pre-pulse protocols under voltage clamp have been used to show that part of this conductance arises from the incompletely blocked delayed rectifier. 6. Substantial reductions in this conductance are produced by anaesthetics at 'clinical' concentrations. 7. It is concluded that there is a component of the K+ conductance of the resting squid axon other than the Hodgkin-Huxley delayed rectifier which is extremely sensitive to anaesthetics and which to an appreciable extent determines the resting potential.

Intracellular calcium transients were recorded from voltage-clamped frog twitch muscle fibres using Arsenazo III. The possible role of extracellular ions in excitation-contraction (e.-c.) coupling was examined using ion substitutions and blocking drugs in the bathing medium. Parameters measured included the Arsenazo response size to a standard depolarizing pulse (5 ms, 0 mV) and the strength-duration curve for threshold Arsenazo signal. Addition of tetrodotoxin (TTX) decreased the response size to small (-30 mV, 5 ms), but not large (+30 mV, 10 ms) depolarizations, probably because of poor voltage clamp of the tubular membrane in the absence of TTX. Clamping TTX-treated fibres with the wave form of a recorded action potential gave an Arsenazo response similar to that elicited by the normal action potential (at 10 degrees C). Complete substitution of sodium (by choline, lithium or Tris) or chloride (by methyl sulphate or maleate) in the bathing solution gave no appreciable changes in the size of the Arsenazo response. Reduction of extracellular free [Ca2+] to low levels using EGTA caused a slight reduction in the calcium signal elicited by the standard depolarization (to 74% after a few hours, and to 62% after 2 days; temperature 5-10 degrees C). The strength-duration curve was unchanged. Arsenazo responses about 75% of the control size could be elicited in high potassium solution (42 mM-K2SO4) by strong (+80 mV, 20 ms) depolarizations, after re-polarizing the fibres to -90 mV for a few minutes. The voltage dependence of activation was shifted to more positive potentials in this solution. Tetraethylammonium (TEA) bromide at a concentration of 20 mM did not alter the Arsenazo signal, whilst 120 mM-TEA reduced the response by 25%. 3,4-diaminopyridine (DAP) reduced the size of the Arsenazo signal at a concentration of 5 mM, and caused spontaneous release of calcium from the sarcoplasmic reticulum (s.r.) in the absence of membrane potential changes. The Arsenazo signal elicited by an action potential was enhanced by 1 mM-DAP, because of prolongation of the action potential, but was depressed by higher concentrations. We conclude that e.-c. coupling does not involve the influx of any external ions into the muscle fibre. If a current flow between the T-tubules and the s.r. is involved in e.-c. coupling, then this is probably carried by an efflux of potassium ions.

Slow action potentials were evoked in twitch fibres of rat extensor digitorum longus (e.d.l.) and soleus muscles after drastically reducing the Cl and K conductances of the muscle fibres. Cl conductance was eliminated by exposing the muscles to a Cl-free saline in which methanesulphonate replaced Cl. K conductance was reduced by adding tetraethylammonium (TEA) and 3,4-diaminopyridine (3,4-DAP) to the Cl-free saline or by overnight incubation of the muscles in a saline containing Cs and TEA. The delayed rectifier was markedly blocked by TEA and 3,4-DAP. In contrast, the inward rectifier was blocked only by TEA. Depolarization with pulses of increasing amplitude triggered slow responses which had a threshold of -30 to -10 mV and a peak amplitude of 50-60 mV. In e.d.l. muscles the time course of the response was sustained for the duration of the pulses and was not affected by repeated stimulation. In soleus muscles the first evoked response was sustained in about 60% of the fibres and transient in the rest. Transient responses reached a peak amplitude and were followed by a hyperpolarization. Repeated stimulation irreversibly transformed the sustained responses of soleus fibres into transient ones. The responses were blocked when the Ca in saline was replaced by Mg (10 mM) or Co (5 mM) or by the addition of Cd (0.1-1.0 mM) or nifedipine (5-6 microM). Tetrodotoxin did not affect the responses. These results strongly suggest that Ca is the main carrier of current during the response. Nifedipine blocked both the Ca response and the subsequent hyperpolarization, suggesting that the latter is due to the activation of a Ca-dependent K conductance.

1. The effects of botulinum toxin (BoTx) types A and D on spontaneous and evoked phasic transmitter release were studied in the isolated extensor digitorum longus muscle of the rat or the levator auris longus muscle of mice. 2. The toxins were injected subcutaneously into the hindleg of adult rats or the dorsal aspect of the neck of mice. At various times after the injection the muscles were removed from the anaesthetized animal and neuromuscular transmission examined in vitro by conventional intracellular techniques. 3. Both toxins reduced spontaneous transmitter release recorded as the frequency of miniature end-plate potentials but BoTx type D was less effective in that respect than the type A toxin. 4. With both toxins the block of evoked phasic transmitter release, recorded as end-plate potentials, was almost complete. As previously reviewed by Simpson (1986) the block produced by BoTx type A was partially reversed by procedures which elevate the intraterminal level of calcium ions. However, in BoTx type D-paralysed muscles such procedures failed to restore phasic transmitter release but caused a period of high-frequency asynchronous transmitter release following each nerve impulse. 5. To investigate if the lack of synchronization of evoked transmitter release observed in BoTx type D-paralysed muscles was due to alterations in presynaptic currents we examined, by perineural recordings, the Na+, fast K+, slow K+, K+-Ca2+-dependent and the Ca2+ currents in BoTx type D-paralysed muscles. These presynaptic currents were not altered as compared to unpoisoned controls. 6. We suggest that there exists a presynaptic process, which in addition to Ca2+ influx participates in transmitter synchronization and which is a main target for BoTx type D action.

1. The m. triangularis sterni of the mouse was used to investigate the actions of dendrotoxin, beta-bungarotoxin, crotoxin, taipoxin, bee venom phospholipase A2, aprotinin and apamin on presynaptic currents which flow inside the perineural sheath of nerve bundles upon nerve stimulation. 2. Neither the fast K+ current (IK,f) nor the Ca2+-dependent K+ current IK(Ca) (unmasked after blockade of IK,f by 3,4-diaminopyridine) was affected by the neurotoxins and drugs mentioned. 3. Inhibition of both IK,f and IK(Ca) by tetraethylammonium (30 mM) prolonged presynaptic depolarization owing to Ca2+ influx through fast and slow Ca2+ channels. Additional application of dendrotoxin, beta-bungarotoxin, crotoxin or taipoxin in the nanomolar range caused further prolongation of Ca2+ influx, presumably due to blockade of slowly activating K+ current (IK,s). Onset of toxin effects was immediate and could not be reversed by washing for 60 min. 4. Similar prolongation of slow Ca2+ current was effected by 3,4-diaminopyridine, whereas addition of apamin, aprotinin or phospholipase A2 left the signals unchanged. 5. These data indicate that facilitatory actions of dendrotoxin, beta-bungarotoxin, taipoxin and crotoxin are mediated by an increase of Ca2+ entry into nerve terminals. The actions of these toxins are discussed in terms of a blockade of presynaptic K+ channels with slow activation kinetics.