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3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 μM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 μM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-μM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-μM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-μM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder caused by antibodies directed against the voltage-gated calcium channels that provide the calcium ion flux that triggers acetylcholine release at the neuromuscular junction. To study the pathophysiology of LEMS and test candidate therapeutic strategies, a passive-transfer animal model has been developed in mice, which can be created by daily intraperitoneal injections of LEMS patient serum or IgG into mice for 2-4 weeks. Results from studies of the mouse neuromuscular junction have revealed that each synapse has hundreds of transmitter release sites but that the probability for release at each one is likely to be low. LEMS further reduces this low probability such that transmission is no longer effective at triggering a muscle contraction. The LEMS-mediated attack reduces the number of presynaptic calcium channels, disorganizes transmitter release sites, and results in the homeostatic upregulation of other calcium channel types. Symptomatic treatment is focused on increasing the probability of release from dysfunctional release sites. Current treatment uses the potassium channel blocker 3,4-diaminopyridine (DAP) to broaden the presynaptic action potential, providing more time for calcium channels to open. Current research is focused on testing new calcium channel gating modifiers that work synergistically with DAP.

Glial cells were originally thought to be fairly passive players in the nervous system, mainly involved in supporting neurons by regulating ion concentrations and other substances in the extracellular fluid. However, it is now clear that glial cells also have a much more direct, integral role in regulating synaptic transmission. In particular, a subset of glial cells called astrocytes often surround synapses and are able to affect synaptic transmission by removing released neurotransmitters from the synaptic cleft, maintaining appropriate ion concentrations in the extracellular fluid, and releasing neurotransmitters themselves in a manner similar to synaptic vesicular release from neurons. Additionally, astrocytes are connected to one another by gap junctions, which allows coordinated modulation of neighboring astrocytes by waves of calcium that propagate via these gap junctions, and, by extension, coordinate the activity of local neurons.

The fusion of synaptic vesicles and release of chemical transmitter from the axon terminal occurs very rapidly (in less than 1 ms), and is dependent upon an influx of calcium ions. What are the biochemical processes that trigger vesicle fusion, and how can they occur so quickly? First, vesicles are moved to the active zone and tethered to the plasma membrane to prepare them for release in advance of action potential activity. Second, a calcium sensor protein on synaptic vesicles holds the vesicle on the verge of fusion, waiting for the calcium ion trigger. Finally, when calcium ions enter the nerve terminal, they bind to the calcium sensor protein, releasing the hold on the synaptic vesicle, which facilitates very rapid fusion with the plasma membrane.

The impact of presynaptic transmitter release site organization on synaptic function has been a vibrant area of research for synaptic physiologists. Because there is a highly nonlinear relationship between presynaptic calcium influx and subsequent neurotransmitter release at synapses, the organization and density of calcium sources (voltage-gated calcium channels [VGCCs]) relative to calcium sensors located on synaptic vesicles is predicted to play a major role in shaping the dynamics of neurotransmitter release at a synapse. Here we review the history of structure-function studies within transmitter release sites at the neuromuscular junction across three model preparations in an effort to discern the relationship between VGCC organization and synaptic function, and whether that organizational structure imparts evolutionary advantages for each species.

Voltage-gated calcium channels (VGCCs) are critical regulators of many cellular functions, including the activity-dependent release of chemical neurotransmitter from nerve terminals. At nerve terminals, the Cav2 family of VGCCs are closely positioned with neurotransmitter-containing synaptic vesicles. The relationship between calcium ions and transmitter release is such that even subtle changes in calcium flux through VGCCs have a strong influence on the magnitude of transmitter released. Therefore, modulators of the calcium influx at nerve terminals have the potential to strongly affect transmitter release at synapses. We have previously developed novel Cav2-selective VGCC gating modifiers (notably GV-58) that slow the deactivation of VGCC current, increasing total calcium ion flux. Here, we describe ten new gating modifiers based on the GV-58 structure that extend our understanding of the structure-activity relationship for this class of molecules and extend the range of modulation of channel activities. In particular, we show that one of these new compounds (MF-06) was more efficacious than GV-58, another (KK-75) acts more quickly on VGCCs than GV-58, and a third (KK-20) has a mix of increased speed and efficacy. A subset of these new VGCC agonist gating modifiers can increase transmitter release during action potentials at neuromuscular synapses, and as such, show potential as therapeutics for diseases with a presynaptic deficit that results in neuromuscular weakness. Further, several of these new compounds can be useful tool compounds for the study of VGCC gating and function.

The frog neuromuscular junction (NMJ) is a strong and reliable synapse because, during activation, sufficient neurotransmitter is released to trigger a postsynaptic action potential (AP). Recent evidence supports the hypothesis that this reliability emerges from the assembly of thousands of unreliable single vesicle release sites. The mechanisms that govern this unreliability include a paucity of voltage-gated calcium channels, a low probability of calcium channel opening during an AP, and the rare triggering of synaptic vesicle fusion even when a calcium channel does open and allows calcium flux. Here, we discuss the evidence that these unreliable single vesicle release sites may be the fundamental building blocks of many types of synapses in both the peripheral and central nervous system (PNS and CNS, respectively).

We used high-resolution fluorescence imaging and single-pixel optical fluctuation analysis to estimate the opening probability of individual voltage-gated calcium (Ca2+) channels during an action potential and the number of such Ca2+channels within active zones of frog neuromuscular junctions. Analysis revealed ∼36 Ca2+channels within each active zone, similar to the number of docked synaptic vesicles but far less than the total number of transmembrane particles reported based on freeze-fracture analysis (∼200–250). The probability that each channel opened during an action potential was only ∼0.2. These results suggest why each active zone averages only one quantal release event during every other action potential, despite a substantial number of docked vesicles. With sparse Ca2+channels and low opening probability, triggering of fusion for each vesicle is primarily controlled by Ca2+influx through individual Ca2+channels. In contrast, the entire synapse is highly reliable because it contains hundreds of active zones.

N-type calcium currents are important in many neuronal functions, including cellular signaling, regulation of gene expression, and triggering of neurotransmitter release. Often the control of these diverse cellular functions is governed by the spatial and temporal patterns of calcium entry in subcellular compartments. Underlying this issue is the effectiveness of action potentials at triggering calcium channel opening. Chick ciliary ganglion neurons were used as model cells to study the activation of N-type calcium current during action potential depolarization. Several different action potential shapes were recorded, used as voltage command templates, and altered such that control action potential–evoked currents could be compared with those elicited by broadened action potential commands. Depending on the action potential shape used to activate calcium currents in chick ciliary ganglion neurons, and the temperature at which recordings were performed, varying proportions ( I/ Imax) of N-type calcium current could be activated. The largest proportion measured occurred using a broad ciliary ganglion cell soma action potential to activate calcium current at 37°C (100%). The smallest proportion measured occurred using a fast, high-temperature–adjusted frog motoneuron nerve terminal action potential to activate calcium current at room temperature (10%). These data are discussed with respect to the impact on cellular signaling and the regulation of transmitter release.

The synapse between a spinal motor neuron and a muscle cell is normally very effective at eliciting muscle contraction. A reliable connection between these two cells occurs because a single action potential reaching the motor nerve terminal normally releases hundreds of packets of transmitter containing thousands of chemical transmitter molecules, which cross the synapse and encounter a specialized region of postsynaptic muscle. Within the muscle membrane are thousands of receptor proteins specific for this transmitter. Activation of these postsynaptic receptors allows positively charged ions to cross the muscle membrane, generating a muscle cell action potential that leads to muscle contraction. Because of its size, contraction of a muscle cell requires the activation of an exceptionally large number of neurotransmitter receptors. To understand the regulation of this reliable communication and to elucidate details of pathological conditions that lead to muscle weakness, we have studied the subcellular mechanisms that govern synaptic transmission at the neuromuscular junction (NMJ). This article will review recent electron microscopic, electrophysiological, and imaging data in a discussion of the function of the motor nerve terminal in both normal and diseased states. Taken together, the existing data lead us to hypothesize that a small fraction of available calcium channels open within the transmitter releasing regions of the NMJ and that each vesicle fusion event is triggered by calcium flux through a single channel opening.

Investigations over the last two decades have made major inroads in clarifying the cellular and molecular events that underlie the fast, synchronous release of neurotransmitter at nerve endings. Thus, appreciable progress has been made in establishing the structural features and biophysical properties of the calcium (Ca2+) channels that mediate the entry into nerve endings of the Ca2+ ions that trigger neurotransmitter release. It is now clear that presynaptic Ca2+ channels are regulated at many levels and the interplay of these regulatory mechanisms is just beginning to be understood. At the same time, many lines of research have converged on the conclusion that members of the synaptotagmin family serve as the primary Ca2+ sensors for the action potential-dependent release of neurotransmitter. This identification of synaptotagmins as the proteins which bind Ca2+ and initiate the exocytotic fusion of synaptic vesicles with the plasma membrane has spurred widespread efforts to reveal molecular details of synaptotagmin's action. Currently, most models propose that synaptotagmin interfaces directly or indirectly with SNARE (soluble, N-ethylmaleimide sensitive factor attachment receptors) proteins to trigger membrane fusion. However, in spite of intensive efforts, the field has not achieved consensus on the mechanism by which synaptotagmins act. Concurrently, the precise sequence of steps underlying SNARE-dependent membrane fusion remains controversial. This review considers the pros and cons of the different models of SNARE-mediated membrane fusion and concludes by discussing a novel proposal in which synaptotagmins might directly elicit membrane fusion without the intervention of SNARE proteins in this final fusion step.

Short-term synaptic facilitation occurs during high-frequency stimulation, is known to be dependent on presynaptic calcium ions, and persists for tens of milliseconds after a presynaptic action potential. We have used the frog neuromuscular junction as a model synapse for both experimental and computer simulation studies aimed at testing various mechanistic hypotheses proposed to underlie short-term synaptic facilitation. Building off our recently reported excess-calcium-binding-site model of synaptic vesicle release at the frog neuromuscular junction (Dittrich M, Pattillo JM, King JD, Cho S, Stiles JR, Meriney SD. Biophys J 104: 2751–2763, 2013), we have investigated several mechanisms of short-term facilitation at the frog neuromuscular junction. Our studies place constraints on previously proposed facilitation mechanisms and conclude that the presence of a second class of calcium sensor proteins distinct from synaptotagmin can explain known properties of facilitation observed at the frog neuromuscular junction. We were further able to identify a novel facilitation mechanism, which relied on the persistent binding of calcium-bound synaptotagmin molecules to lipids of the presynaptic membrane. In a real physiological context, both mechanisms identified in our study (and perhaps others) may act simultaneously to cause the experimentally observed facilitation. In summary, using a combination of computer simulations and physiological recordings, we have developed a stochastic computer model of synaptic transmission at the frog neuromuscular junction, which sheds light on the facilitation mechanisms in this model synapse.

1. The voltage dependence and kinetic properties of stage 40 ciliary ganglion calcium currents were determined using short (10 ms) voltage steps. These properties aided the interpretation of the action potential-evoked calcium current described below, and the comparison of our data with those observed in other preparations. 2. Three different natural action potential waveforms were modelled by a series of ramps to generate voltage clamp commands. Calcium currents evoked by these model action potentials were compared before and after alterations in the repolarization phase of each action potential. 3. Abrupt step repolarizations from various time points were used to estimate the time course of calcium current activation during each action potential. Calcium current evoked by fast action potentials (duration at half-amplitude, 0.5 or 1.0 ms) did not reach maximal activation until the action potential had repolarized by 40-50 %. In contrast, calcium current evoked by a slow action potential (duration at half-amplitude, 2.2 ms) was maximally activated near the peak of the action potential. 4. Slowing the rate of repolarization of the action potential (broadening) from different times was used to examine effects on peak and total calcium influx. With all three waveforms tested, broadening consistently increased total calcium influx (integral). However, peak calcium current was either increased or decreased depending on the duration of the control action potential tested and the specific timing of the initiation of broadening the repolarization phase. 5. The opposite effects on peak calcium current observed with action potential broadening beginning at different time points in repolarization may provide a mechanism for the variable effects of potassium channel blockers on transmitter release magnitude.

Voltage-dependent calcium (Ca2+) currents were characterized and modulatory effects of somatostatin were measured in acutely dissociated chick ciliary ganglion neurons at embryonic stages 34, 37, and 40. This developmental time period coincides with the period of synapse formation between ciliary ganglion neurons and peripheral eye muscles. At all three developmental stages Ca2+ current could be blocked almost completely by combined application of omega-CgTX GVIA and nitrendipine. At young embryonic ages there was significant overlap in sensitivity, with approximately 75% of the current sensitive to either blocker applied independently. By stage 40, there was very little or no overlap in sensitivity, with approximately 75% of the current blocked by omega-CgTX GVIA (N-type) and 30% blocked by nitrendipine (L-type). These data are consistent with earlier findings that the pharmacology of acetylcholine release from ciliary ganglion nerve terminals changes during development from sensitivity to both dihydropyridines and omega-CgTX GVIA to selective sensitivity to omega-CgTX GVIA (Gray et al., 1992). Somatostatin reduced Ca2+ current by 50-60% at all three developmental stages. At early developmental stages somatostatin receptors coupled predominantly to the current that was sensitive to both omega-CgTX GVIA and nitrendipine. By stage 40, somatostatin primarily inhibited classically defined N-type current (selectively sensitive to omega-CgTX GVIA). Thus, somatostatin receptor coupling to Ca2+ channels persisted throughout development as Ca2+ current pharmacology changed.

We developed a novel calcium (Ca(2+)) channel agonist that is selective for N- and P/Q-type Ca(2+) channels, which are the Ca(2+) channels that regulate transmitter release at most synapses. We have shown that this new molecule (GV-58) slows the deactivation of channels, resulting in a large increase in presynaptic Ca(2+) entry during activity. GV-58 was developed as a modification of (R)-roscovitine, which was previously shown to be a Ca(2+) channel agonist, in addition to its known cyclin-dependent kinase activity. In comparison with the parent molecule, (R)-roscovitine, GV-58 has a ∼20-fold less potent cyclin-dependent kinase antagonist effect, a ∼3- to 4-fold more potent Ca(2+) channel agonist effect, and ∼4-fold higher efficacy as a Ca(2+) channel agonist. We have further evaluated GV-58 in a passive transfer mouse model of Lambert-Eaton myasthenic syndrome and have shown that weakened Lambert-Eaton myasthenic syndrome-model neuromuscular synapses are significantly strengthened following exposure to GV-58. This new Ca(2+) channel agonist has potential as a lead compound in the development of new therapeutic approaches to a variety of disorders that result in neuromuscular weakness.

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca(2+)-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20-40 independent Ca(2+)-binding sites on synaptic vesicles, only a fraction of which need to bind Ca(2+) to trigger fusion, are sufficient to predict physiological release. Our excess-Ca(2+)-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca(2+)-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca(2+) dynamics during synapse activation.

Transmitter release from frog motor nerve terminals occurs at specialized sites on the nerve terminal called active zones (AZs). We have used a low calcium (0.1 nM) saline treatment to disrupt AZ structure and correlated these changes with alterations in transmitter release from the nerve terminal. Exposure to 0.1 nM free calcium saline for 3 h caused many individual AZs to break into two or three pieces, apparently unorganized particles drifted free of the AZ array, and the normally ordered alignment of AZ particles was loosened. Despite these forms of disruption in AZ organization, physiological function remained remarkably normal. Although the size of the endplate potential recorded in response to a single nerve stimulus was little affected, paired-pulse facilitation and tetanic potentiation were significantly increased. Synaptic depression was not apparent during the tetanus, but was revealed following the cessation of the stimulation. The results are consistent with the hypothesis that 0.1 nM calcium treatment detached AZ segments from the anchoring molecules that normally hold these proteins in alignment with other synapse-specific molecules. We propose that the ordered AZ organization serves to bring the calcium channels that regulate transmitter release in close proximity to other proteins that are critical to the modulation of release, especially during periods of high frequency stimulation. We hypothesize that the drifting AZ segments, although capable of apparently normal transmitter release, may not be tightly coupled with the intracellular calcium handling proteins that normally restrict the time that calcium ions have to act on the transmitter release apparatus following each action potential.

The signal transduction cascade between the activation of the somatostatin (SOM) receptor and modulation of transmitter release was study using Acetylcholine (Ach) release measurements and patch clamp recordings of Ca2+ current from acutely dissociated St 40 ciliary ganglion neurons. As in intact synapses, somal ACh release was blocked by 100 nM SOM or 100 microM dibutyril cGMP, and the SOM-mediated inhibition could be reversed by 10 microM 1-NAME (a selective inhibitor of nitric oxide synthase, NOS) or 100 microM Rp-8p-CPT-cGMPs (a selective inhibitor of a cGMP protein dependent kinase, PKG). In whole cell recordings, SOM inhibition of Ca2+ current rapidly relaxes to control levels but is sustained in perforated patch recordings which decreases cell dialysis. Inhibition of NOS or PKG in perforated patch recordings, however caused SOM effects to become transient again. We hypothesize that PKG alters the characteristics of the membrane-delimited G protein inhibition of Ca2+ current. Therefore SOM receptors trigger a membrane-delimited signal transduction cascade that is modulated by soluble messengers, converging on voltage activated Ca2+ channels. When both pathways are active together, SOM causes a sustained inhibition of neuronal Ca2+ current leading to a decrease in transmitter release.

Dynasore was recently developed as a small molecule, selective non-competitive inhibitor of the protein dynamin. This inhibitor has been shown to block dynamin-dependent endocytosis and is now used commonly to study vesicular recycling at synapses. We have measured the effects of dynasore on spontaneous and evoked transmitter release at the frog neuromuscular junction and shown that, in addition to inhibiting endocytosis, dynasore increases the probability of transmitter release. Furthermore, we have shown that dynasore exposure leads to an increase in resting intra-terminal calcium, but this effect does not completely account for the dynasore-mediated increase in the probability of transmitter release. Therefore, in interpreting effects of the dynamin inhibitor dynasore at synapses, one must be alert to potential increases in presynaptic calcium concentration and transmitter release probability.

The nature of presynaptic calcium (Ca2+) signals that initiate neurotransmitter release makes these signals difficult to study, in part because of the small size of specialized active zones within most nerve terminals. Using the frog motor nerve terminal, which contains especially large active zones, we show that increases in intracellular Ca2+concentration within 1 msec of action potential invasion are attributable to Ca2+entry through N-type Ca2+channels and are not uniformly distributed throughout active zone regions. Furthermore, changes in the location and magnitude of Ca2+signals recorded before and after experimental manipulations (ω-conotoxin GVIA, diaminopyridine, and lowered extracellular Ca2+) support the hypothesis that there is a remarkably low probability of a single Ca2+channel opening within an active zone after an action potential. The trial-to-trial variability observed in the spatial distribution of presynaptic Ca2+entry also supports this conclusion, which differs from the conclusions of previous work in other synapses.

Using Xenopus nerve-muscle co-cultures, we have examined the contribution of calcium-activated potassium (KCa) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of KCa channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolar- ization at the active zone caused by potassium current through the KCa channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium influx was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents. From these data, we suggest that the large-conductance KCa channels expressed at presynaptic varicosities regulate transmitter release magnitude during single action potentials by altering the rate of action potential repolarization, and thus the magnitude of peak calcium current. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

We have studied voltage-dependent inhibition of N-type calcium currents to investigate the effects of G-protein modulation-induced alterations in channel gating on action potential-evoked calcium current. In isolated chick ciliary ganglion neurons, GTPγS produced voltage-dependent inhibition that exhibited slowed activation kinetics and was partially relieved by a conditioning prepulse. Using step depolarizations to evoke calcium current, we measured tail current amplitudes on abrupt repolarization to estimate the time course of calcium channel activation from 1 to 30 ms. GTPγS prolonged significantly channel activation, consistent with the presence of kinetic slowing in the modulated whole cell current evoked by 100-ms steps. Since kinetic slowing is caused by an altered voltage dependence of channel activation (such that channels require stronger or longer duration depolarization to open), we asked if GTPγS-induced modulation would alter the time course of calcium channel activation during an action potential. Using an action potential waveform as a voltage command to evoke calcium current, we abruptly repolarized to −80 mV at various time points during the repolarization phase of the action potential. The resulting tail current was used to estimate the relative number of calcium channels that were open. Using action potential waveforms of either 2.2- or 6-ms duration at half-amplitude, there were no differences in the time course of calcium channel activation, or in the percent activation at any time point tested during the repolarization, when control and modulated currents were compared. It is also possible that modulated channels might open briefly and that these reluctant openings would effect the time course of action potential-evoked calcium current. However, when control and modulated currents were scaled to the same peak amplitude and superimposed, there was no difference in the kinetics of the two currents. Thus voltage-dependent inhibition did not alter the kinetics of action potential-evoked current. These results suggest that G-protein-modulated channels do not contribute significantly to calcium current evoked by a single action potential.

The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonicXenopusneuromuscular junctions and simultaneous pre- and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (KCa) currents. The onset ofKCacurrent preceded the peak of the action potential. The predominantly ω-CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca2+entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current. ω-CgTX also blockedKCacurrents and transmitter release during an action potential, suggesting that Ca andKCachannels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments,KCachannel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation ofKCachannels or the diffusion/removal of Ca2+from sites of neurotransmitter release after an action potential.

Barium currents through voltage-gated calcium (Ca2+) channels were studied in the small-cell lung carcinoma cell line NCI-H345 using patch clamp techniques. Pharmacological dissection of whole-cell barium currents revealed that 23% of the current was sensitive to nitrendipine, 35% to omega-conotoxin GVIA, and between 10 and 39% to omega-Aga-IVA. This implies that these cells express L-, N-, and P-type calcium channels. Only large cells expressed current that was sensitive to omega-Aga-IVA. The size dependency of this P-type channel expression may reflect the cell cycle stage. Cell-attached recordings revealed three unitary conductances: 5 to 6 pS, 10 to 12 pS, and 20 to 23 pS. The largest conductance channel (20-23 pS) was sensitive to Bay K 8644 and is presumed to represent L-type calcium channels. The frequency of observing the medium conductance channel (10-12 pS) was reduced by exposure to omega-conotoxin GVIA and may represent N-type channels. Incubation of cells with Lambert-Eaton myasthenic syndrome IgG for 24 to 48 hours removed up to 71% of the whole-cell current. Incubation with control human IgG (normal or myasthenia gravis) had no effect. Lambert-Eaton myasthenic syndrome IgG did not selectively target one "presynaptic" type of calcium channel, but rather appeared to target many of the calcium channel types that are expressed on small-cell lung carcinoma cells.

Neurotransmitter release is frequently regulated by peptides that modulate neuronal calcium channels. Whole-cell recordings show that the ion permeability and voltage dependence of these channels are controlled by a membrane-associated pathway involving GTP-binding proteins. Here we use perforated-patch recordings to show that, in addition to this pathway, the peptide somatostatin inhibits the calcium current in chick ciliary ganglion neurons by a second soluble pathway involving a cyclic GMP-dependent protein kinase (cGMP-PK). This somatostatin inhibition of Ca2+ current did not desensitize and was not characterized by the slowing of Ca(2+)-current activation (kinetic slowing) observed in whole-cell recordings. When cGMP-PK was inhibited, somatostatin inhibition of Ca2+ current resembled that observed with whole-cell recordings. cGMP agonists mimic the effect of somatostatin only in perforated patch recordings. An endogenous cGMP-PK therefore forms part of the mechanism by which somatostatin induces a sustained inhibition of neuronal calcium channels.