Protein kinase Calpha modulates depolarizaton-evoked changes of intracellular Ca2+ concentration in a rat pheochromocytoma cell line.

Conventional protein kinase C (cPKC) isoforms are activated by a coincident rise in cytosolic Ca(2+) and membrane-bound diacylglycerol. In excitable cells, cPKC may be activated by Ca(2+) influx through voltage-gated Ca(2+) channels (VGCC). cPKCs, in turn, are known to modulate the activity of VGCC. We examined whether PKCalpha, a cPKC, could be activated by depolarization in a neuroendocrine cell line and whether activation occurred on a time scale that modulated the depolarization-evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) signal. Pheochromocytoma cells (PC12 cells) were transfected with wild-type and mutant forms of PKCalpha labeled with yellow fluorescent protein to monitor kinase translocation. Simultaneously, [Ca(2+)](i) changes were monitored with fura-2. Two point mutations that render PKCalpha inactive, D187A in the Ca(2+) binding site and K368R in the ATP binding site, significantly prolonged the time-to-peak of the depolarization-evoked [Ca(2+)](i) signal. A mutation that modulates membrane insertion (W58G) and two mutations of an autophosphorylation site (S657A, S657E) had no effect on the kinetics of the [Ca(2+)](i) signal. We conclude that in PC12 cells, Ca(2+) entry through VGCC rapidly activates PKCalpha, and that PKCalpha can modulate the Ca(2+) signal on a physiologically relevant time scale. Point mutations of PKCalpha can be used as specific and potent modulators of the PKC signaling pathway.

A current activated on depletion of intracellular Ca2+ stores can regulate exocytosis in adrenal chromaffin cells.

Exocytosis in excitable cells is strongly coupled to Ca2+ entry through voltage-gated channels but can be evoked by activation of membrane receptors that release Ca2+ from inositol 1,4, 5-trisphosphate-sensitive internal stores. In many cell types, depletion of Ca2+ stores activates Ca2+ influx across the plasma membrane, a process known as capacitative or store-operated Ca2+ entry. This influx is mediated by a number of voltage-independent, Ca2+-selective currents. In addition to replenishing Ca2+ stores, these currents are hypothesized to play an important role in agonist-evoked secretion in nonexcitable cells, although this has not been confirmed experimentally. The existence and physiological function of such currents in excitable cells is not known. Using the capacitance detection technique to monitor exocytosis, we provide direct experimental evidence that a similar mechanism exists in bovine adrenal chromaffin cells. Depletion of intracellular Ca2+ stores with thapsigargin, a SERCA pump inhibitor, or with BAPTA, an exogenous Ca2+ chelator, activates a small-amplitude, voltage-independent current that is carried by Ca2+ and Na+ ions. Ca2+ entry through this pathway is sufficient to stimulate exocytosis at negative membrane potentials. In addition, depolarization-evoked exocytosis is markedly facilitated on activation of the current. These data suggest that excitable cells possess a store-operated Ca2+ influx mechanism that may both directly trigger exocytosis and modulate excitation-secretion coupling.

Lambert-Eaton antibodies inhibit Ca2+ currents but paradoxically increase exocytosis during stimulus trains in bovine adrenal chromaffin cells.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that affects neurotransmitter release at peripheral synapses. LEMS antibodies inhibit Ca2+ currents in excitable cells, but it is not known whether there are additional effects on stimulus-secretion coupling. The effect of LEMS antibodies on Ca2+ currents and exocytosis was studied in bovine adrenal chromaffin cells using whole-cell voltage clamp in perforated-patch recordings. Purified LEMS IgGs from five patients inhibited N- and P/Q-type Ca2+ current components to different extents. The reduction in Ca2+ current resulted in smaller exocytotic responses to single depolarizing pulses, but the normal relationship between integrated Ca2+ entry and exocytosis (Enisch and Nowycky, 1996) was preserved. The hallmark of LEMS is a large potentiation of neuromuscular transmission after high-frequency stimulation. In chromaffin cells, stimulus trains can induce activity-dependent enhancement of the Ca2+-exocytosis relationship. Enhancement during trains occurs most frequently when pulses are brief and evoke very small amounts of Ca2+ entry (Engisch et al., 1997). LEMS antibody treatment increased the percentage of trains eliciting enhancement through two mechanisms: (1) by reducing Ca2+ entry and (2) through a Ca2+-independent effect on the process of enhancement. This leads to a paradoxical increase in the amount of exocytosis during stimulus trains, despite inhibition of Ca2+ currents.

Excitation-secretion coupling in mammalian neurohypophysial nerve terminals.

1. Oxytocin and vasopressin secretion from the neurohypophysis (NHP) is evoked by strongly patterned bursts of action potentials. We studied excitation-secretion coupling in single isolated terminals of rat NHP using patch clamp and capacitance detection techniques. 2. The secretory response evoked by trains of depolarizing pulses consisted of two discrete phases. Ca2+ entry during pulses early in the train did not elicit secretion. Exocytotic responses began only after a characteristic amount of total Ca2+ entry called "threshold". 3. In the postthreshold secretory phase, exocytotic events occurred during or immediately after depolarizing pulses, indicating that the final Ca(2+)-dependent step is triggered by high Ca2+ concentrations near the plasma membrane that dissipate rapidly after channel closure. Secretion was sensitive to both the concentration and species of Ca2+ chelator. BAPTA, a Ca2+ chelator with rapid Ca2+ binding kinetics, was more effective than EGTA in diminishing secretion. 4. The "threshold" amount of Ca2+ was determined by the concentration, but not species, of Ca2+ chelator. The threshold value was constant even when Ca2+ entry parameters were varied over a broad range of current amplitudes, pulse durations, and number of pulses, indicating that it did not require high Ca2+ concentrations near the plasma membrane. 5. These results suggest that the secretory response to a train of pulses consists of a Ca(2+)-dependent preparatory step that must be completed before subsequent Ca2+ entry can elicit exocytosis. 6. Exocytotic responses during single trains showed strong depression at a step subsequent to Ca2+ entry. Recovery from depression required 30-60 sec. 7. The properties of threshold secretion observed in NHP terminals are discussed in terms of current models of secretion.

Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromaffin cells.

1. Endocytosis following exocytosis evoked by single step depolarizations was examined in bovine adrenal chromaffin cells using high resolution capacitance measurements in perforated-patch voltage clamp recordings. 2. Endocytosis was detected as a smooth exponential decline in membrane capacitance to either the pre-stimulus level ('compensatory retrieval') or far below the pre-stimulus level ('excess retrieval'). During excess retrieval, > 10% of the cell surface could be internalized in under 5 s. 3. Compensatory retrieval was equal in magnitude to stimulus-evoked exocytosis for membrane additions > 100 fF (about fifty large dense-cored vesicles). In contrast, excess retrieval surpassed both the stimulus-evoked exocytosis, and the initial capacitance level recorded at the onset of phase-tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly returned to pre-stimulus levels, even in the absence of stimulation. 4. A large percentage of capacitance increases < 100 fF, usually evoked by 40 ms depolarizations, were not accompanied by membrane retrieval. 5. Compensatory retrieval could occur with any amount of Ca2+ entry, but excess retrieval was never triggered below a threshold Ca2+ current integral of 70 pC. 6. The kinetics of compensatory and excess retrieval differed by an order of magnitude. Compensatory retrieval was usually fitted with a single exponential function that had a median time constant of 5.7 s. Excess retrieval usually occurred with double exponential kinetics that had an extremely fast first time constant (median, 670 ms) and a second time constant indistinguishable from that of compensatory retrieval. 7. The speed of compensatory retrieval was Ca2+ dependent: the largest mono-exponential time constants occurred for the smallest amounts of Ca2+ entry and decreased with increasing Ca2+ entry. The Ca2+ dependence of mono-exponential time constants was disrupted by cyclosporin A (CsA), an inhibitor of the Ca(2+)- and calmodulin-dependent phosphatase calcineurin. 8. CsA also reduced the proportion of responses with excess retrieval, but this action was caused by a shift in Ca2+ entry values below the threshold for activation. The lower total Ca2+ entry in the presence of CsA was due to an increase in the rate of Ca2+ current inactivation rather than a reduction in peak amplitude. 9. Our data suggest that compensatory and excess retrieval represent two independent, Ca(2+)-regulated mechanisms of rapid membrane internalization in bovine adrenal chromaffin cells. Alternatively, there is a single membrane internalization mechanism that can switch between two distinct modes of behaviour.

Short-term changes in the Ca2+-exocytosis relationship during repetitive pulse protocols in bovine adrenal chromaffin cells.

Stimulus-secretion coupling was monitored with capacitance detection in bovine chromaffin cells recorded in perforated patch mode and stimulated with trains of depolarizing pulses. A subset of stimulus trains evoked a response with a Ca2+-exocytosis relationship identical to that obtained for single depolarizing pulses (Engisch and Nowycky, 1996). Other trains evoked responses with enhanced or diminished Ca2+ efficacy relative to this input-output function. The probability of obtaining a particular Ca2+-exocytosis relationship was correlated with the amount of Ca2+ entry per pulse, such that shorter pulses or smaller currents were associated with the greatest efficacy, and longer pulses and larger currents with the lowest efficacy. Apparent enhancements in Ca2+ efficacy were not caused by residual Ca2+ summing between pulses, because decreasing the interval between pulses usually reduced efficacy in the same cell; conversely, increasing the interval between pulses did not prevent an enhanced Ca2+-exocytosis relationship. Apparent decreases in Ca2+ efficacy were not caused by depletion of an available pool of release-ready vesicles, because an equivalent amount of total Ca2+ entry during a single long depolarizing pulse usually evoked a much larger secretory response in the same cell. Finally, there were no striking differences in global Ca2+ levels monitored with the fluorescent indicator Fura Red that could account for apparent changes in Ca2+ efficacy during repetitive stimulus protocols. It appears that in chromaffin cells, the Ca2+-exocytosis relationship is subject to activity-dependent changes during a stimulus train and can be modulated up or down from a basal state accessed by single pulse stimulations.

Calcium dependence of large dense-cored vesicle exocytosis evoked by calcium influx in bovine adrenal chromaffin cells.

We used the perforated-patch technique to examine the relationship between Ca2+ entry and exocytosis of large dense-cored vesicles in bovine adrenal chromaffin cells. Exocytosis evoked by single-step depolarizations was monitored by capacitance detection. Ca2+ entry was varied by changing external calcium concentration, stepping to different test potentials, depolarizing for different durations, or applying blockers of specific calcium channel subtypes. Regardless of protocol, the amount of exocytosis was strictly related to the integral of the voltage-clamped calcium current, raised to a power of approximately 1.5. Thus, despite the complexities of transient and nonuniform changes in submembrane calcium concentration produced by voltage-gated calcium entry, the calcium dependence of large dense-cored vesicle fusion under conditions of minimal stimulation is well approximated by a simple transfer function of summed calcium entry.

Ba2+ ions evoke two kinetically distinct patterns of exocytosis in chromaffin cells, but not in neurohypophysial nerve terminals.

The coupling between divalent cations and exocytosis of large dense-cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are "stimulus-coupled" to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus-coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20-50 sec. "Stimulus-decoupled" exocytosis is slow (approximately 25-40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5-12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.

Kinetics of stimulus-coupled secretion in dialyzed bovine chromaffin cells in response to trains of depolarizing pulses.

Stimulus-secretion coupling in bovine chromaffin cells was investigated with whole-cell patch-clamp recordings and capacitance detection techniques to monitor exocytosis in response to trains of depolarizing pulses. Two kinetically discrete modes of exocytotic responses were observed. In one mode, the first depolarization of a train elicited a large increase in membrane capacitance (Cm; mean approximately 70 fF). This secretory mode was characterized by small Ca2+ requirements, relative insensitivity to the pipette Ca2+ chelator concentration, and rapid depletion of the secretory response. This mode of stimulus-secretion coupling was labile and was seen only in response to the first and, occasionally, the second stimulus train of whole-cell recordings. The second type of exocytotic response persisted for the remainder of the whole-cell recordings and consisted of two distinct phases. During the earliest pulses of a stimulus train, Ca2+ entry did not evoke Cm increases. Instead, Cm responses were elicited by later pulses, despite diminished Ca2+ entry per pulse caused by Ca2+ channel inactivation. The secretory phase was initiated after a specific "threshold" amount of Ca2+ had entered the cell, which was determined by the concentration, but not the binding kinetics, of the Ca2+ chelator in the pipette. In both the early and the secretory phases, the response of the cell was proportional to cumulative Ca2+ entry, regardless of current amplitude, pulse duration, or number of pulses. Threshold-type secretory kinetics has been described previously in peptide-secreting neurohypophysial (NHP) nerve terminals (Seward et al., 1995). Secretory kinetics with minimal Ca2+ requirements has not been observed in that preparation. Chromaffin cells appear to possess a broader repertoire of stimulus-secretion coupling modes than NHP terminals.

Exocytosis in peptidergic nerve terminals exhibits two calcium-sensitive phases during pulsatile calcium entry.

The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a "shell" rather than "microdomain" model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two-step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.

Activation of nicotinic receptors triggers exocytosis from bovine chromaffin cells in the absence of membrane depolarization.

The traditional function of neurotransmitter-gated ion channels is to induce rapid changes in electrical activity. Channels that are Ca(2+)-permeable, such as N-methyl-D-aspartate receptors at depolarized membrane potentials, can have a broader repertoire of consequences, including changes in synaptic efficacy, developmental plasticity, and excitotoxicity. Neuronal nicotinic receptors for acetylcholine (nAChRs) are usually less Ca(2+)-permeable than N-methyl-D-aspartate receptors but have a significant Ca2+ permeability, which is greater at negative potentials. Here we report that in neuroendocrine cells, activation of nAChRs can trigger exocytosis at hyperpolarized potentials. We used whole-cell patch-clamp recordings to record currents and the capacitance detection technique to monitor exocytosis in isolated bovine chromaffin cells. Stimulation of nAChRs at hyperpolarized potentials (-60 or -90 mV) evokes a large current and a maximal capacitance increase corresponding to the fusion of approximately 200 large dense-core vesicles. The amount of exocytosis is controlled both by the Ca2+ influx through nAChRs and by a contribution from thapsigargin-sensitive Ca2+ sequestering stores. This is a form of neurotransmitter action in which activation of nAChRs triggers secretion through an additional coupling pathway that coexists with classical voltage-dependent Ca2+ entry.

Time courses of calcium and calcium-bound buffers following calcium influx in a model cell.

Fixed and diffusible calcium (Ca) buffers shape the spatial and temporal distribution of free Ca following Ca entry through voltage-gated ion channels. This modeling study explores intracellular Ca levels achieved near the membrane and in deeper locations following typical Ca currents obtained with patch clamp experiments. Ca ion diffusion sets an upper limit on the maximal average Ca concentration achieved near the membrane. Fixed buffers restrict Ca elevation spatially to the outermost areas of the cell and slow Ca equilibration. Fixed buffer bound with Ca near the membrane can act as Ca source after termination of Ca influx. The relative contribution of fixed versus diffusible buffers to shaping the Ca transient is determined to a large extent by the binding rate of each buffer, with diffusible buffer dominating at equal binding rates. In the presence of fixed buffers, diffusible buffers speed Ca equilibration throughout the cell. The concentration profile of Ca-bound diffusible buffer differs from the concentration profile of free Ca, reflecting theoretical limits on the temporal resolution which can be achieved with commonly used diffusible Ca indicators. A Ca indicator which is fixed to an intracellular component might more accurately report local Ca concentrations.

The proton-activated inward current of rat sensory neurons includes a calcium component.

Many neurons possess a proton-activated conductance, IH, which supports a large transient inward current at negative potentials and thereby depolarizes cells during rapid drops in external pH. The channels underlying this conductance are permeant to monovalent cations, with a clear preference for sodium. In earlier experiments, it appeared that divalent cations were impermeant: increasing concentrations of extracellular Ca2+ actually decreased the current amplitude. Using whole-cell patch clamp recording techniques, we find that the proton-activated channel is permeant to Ca2+ ions. In the absence of monovalent cations, a substantial current is supported by divalent cations. The previously reported block results from competition between divalents and monovalents. This finding suggests that IH may provide a pathway for Ca2+ entry during the acidification that accompanies normal synaptic transmission, excessive electrical activity, and tissue ischemia.

Direct measurement of exocytosis and calcium currents in single vertebrate nerve terminals.

The release of neurohormone is widely thought to be exocytotic, involving Ca2(+)-dependent fusion of secretory vesicles with the plasma membrane. The inaccessibility of most nerve ending has so far hampered direct time-resolved measurements of neuronal exocytosis in response to brief depolarization. By using 'whole-terminal' patch-clamp and circuit-analysis techniques to measure membrane capacitance, we have now monitored changes in the surface membrane area of individual nerve terminals isolated from the mammalian neurohypophysis. A single depolarizing pulse leading to Ca2+ entry through voltage-gated calcium channels, rapidly and reproducibly increases the membrane area by an amount corresponding to the fusion of 1-100 secretory vesicles. The magnitude of the capacitance increase depends not only on Ca2+ entry and buffering, but also on the pattern of stimulation revealing facilitation, fatigue and recovery of the release process.

Hippocampal synaptic plasticity induced by excitatory amino acids includes changes in sensitivity to the calcium channel blocker, omega-conotoxin.

The hippocampus is widely used in investigations of different forms of synaptic plasticity, including long-term potentiation and kindling. Receptors for excitatory amino acids (EAAs) play a prominent role in these phenomena. Recently, is has been demonstrated that exposure of hippocampal slices to EAAs and related agonists produces biphasic effects on excitatory synaptic transmission: initial blockade of synaptic responses is followed by a delayed recovery. The recovered responses demonstrate altered pharmacological properties: they acquire sensitivity to N-methyl-D-aspartate (NMDA) antagonists during L-glutamate (Glu) exposure and lose sensitivity to both NMDA and non-NMDA antagonists under L-aspartate (Asp). These changes persist for many hours. It was suggested that this form of hippocampal plasticity may involve transitions between distinct states of synaptic functioning. To explore this possibility, we investigated several properties of synaptic transmission in the initial and EAA-modified states. Here we report that hippocampal postsynaptic potentials (PSPs) evoked under Glu or Asp exposure completely lose sensitivity to omega-conotoxin GVIA (omega-CgTX), a potent, specific, and irreversible blocker of certain types of neuronal calcium channels. After washout of the EAA, sensitivity to the toxin is regained. These results indicate that prolonged EAA exposure induces profound changes in the machinery of synaptic transmission, which include, but are not limited to, changes in calcium channel functioning.

Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals.

The properties of the Ca2+ channels mediating transmitter release in vertebrate neurons have not yet been described with voltage-clamp techniques. Several types of voltage-dependent Ca2+ channels are known to exist on neuronal somata, but the small size and inaccessibility of most vertebrate nerve endings have precluded direct characterization of the presynaptic channels. However, large nerve endings, which release the peptides oxytocin and vasopressin in a Ca2(+)-dependent manner, can be dissociated from the rat neurohypophysis. Using both single-channel and whole-cell patch-clamp techniques, we have characterized two types of Ca2+ channels that coexist in these terminals. One is a large-conductance, high-threshold, dihydropyridine-sensitive channel that contributes a slowly inactivating current. The second is a smaller conductance channel, which is also activated at high thresholds, but underlies a rapidly inactivating, dihydropyridine-insensitive current. Both types of Ca2+ channels may participate in the peptide release process.

Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones.

1. Calcium currents in cultured dorsal root ganglion (d.r.g.) cells were studied with the whole-cell patch-clamp technique. Using experimental conditions that suppressed Na+ and K+ currents, and 3-10 mM-external Ca2+ or Ba2+, we distinguished three distinct types of calcium currents (L, T and N) on the basis of voltage-dependent kinetics and pharmacology. 2. Component L activates at relatively positive test potentials (t.p. greater than -10 mV) and shows little inactivation during a 200 ms depolarization. It is completely reprimed at a holding potential (h.p.) of -60 mV, and can be isolated by using a more depolarized h.p. (-40 mV) to inactivate the other two types of calcium currents. 3. Component T can be seen in isolation with weak test pulses. It begins activating at potentials more positive than -70 mV and inactivates quickly and completely during a maintained depolarization (time constant, tau approximately 20-50 ms). The current amplitude and the rate of decay increase with stronger depolarizations until both reach a maximum at approximately -40 mV. Inactivation is complete at h.p. greater than -60 mV and is progressively removed between -60 and -95 mV. 4. Component N activates at relatively strong depolarizations (t.p. greater than -20 mV) and decays with time constants ranging from 50 to 110 ms. Inactivation is removed over a very broad range of holding potentials (h.p. between -40 and -110 mV). 5. With 10 mM-EGTA in the pipette solution, substitution of Ba2+ for Ca2+ as the charge carrier does not alter the rates of activation or relaxation of any component. However, T-type channels are approximately equally permeable to Ca2+ and Ba2+, while L-type and N-type channels are both much more permeable to Ba2+. 6. Component N cannot be explained by current-dependent inactivation of L current resulting from recruitment of extra L-type channels at negative holding potentials: raising the external Ba2+ concentration to 110 mM greatly increases the amplitude of L current evoked from h.p. = -30 mV but produces little inactivation. 7. Cadmium ions (20-50 microM) virtually eliminate both N and L currents (greater than 90% block) but leave T relatively unaffected (less than 50% block). 200 microM-Cd2+ blocks all three components. 8. Nickel ions (100 microM) strongly reduce T current but leave N and L current little changed. 9. The dihydropyridine antagonist nifedipine (10 microM) inhibits L current (approximately 60% block) at a holding potential that inactivates half the L-type channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Single-channel recordings of three types of calcium channels in chick sensory neurones.

1. T-, and L-type Ca2+ channels were studied in cell-attached patch recordings from the cell bodies of chick dorsal root ganglion neurones. All experiments were performed with isotonic BaCl2 (110 mM) in the recording pipette and with isotonic potassium aspartate in the bathing solution to zero the cell membrane potential. 2. L-type channels are distinguished by a unitary slope conductance of 25 pS, activation over the range of membrane potentials between 0 and +40 mV, little inactivation over the course of a 136 ms depolarization, and availability for opening even at depolarized holding potentials (h.p. greater than -40 mV). L channels show a predominant mode of gating (mode 1) characterized by brief openings (approximately 1 ms), occasionally interspersed with another pattern of gating characterized by much longer openings (mode 2). 3. The dihydropyridine (DHP) Ca2+ agonist Bay K 8644 promotes mode 2 activity and shifts the voltage dependence of L-type channel activation towards more negative potentials. It leaves the unitary current-voltage relation unchanged. 4. Nifedipine, a DHP Ca2+ antagonist, strongly inhibits L-type channel activity through an increase in the proportion of blank sweeps. 5. T-type Ca2+ channels are distinguished by a much smaller unitary slope conductance (8 pS) and by activation and inactivation over relatively negative ranges of potential. Inactivation is complete by the end of 136 ms pulses to test potentials beyond -20 mV. 6. N-type Ca2+ channels are distinguished by an intermediate unitary slope conductance (13 pS), and by activation over a range of potentials between those of T- and L-type channels. Inactivation of N-type channels takes place over an exceptionally broad range of holding potentials (-80 to -20 mV). 7. Cell-attached patch data on the voltage dependence of activation and inactivation of T- and N-type channels are in excellent agreement with results from whole-cell recordings (Fox, Nowycky & Tsien, 1987) if allowances are made for variations in external surface potential. 8. Patches containing one or two channels of a single type were used for analysis of gating kinetics. The predominant pattern of activity for each of the channel types is an exponential distribution of relatively brief (approximately 1 ms) openings, and a bi-exponential distribution of short and long closings. 9. Patches containing all possible combinations of channel types were observed. However, preliminary evidence suggests that channels are distributed unevenly over the cell body; clustering of N-type channels is particularly prominent.(ABSTRACT TRUNCATED AT 400 WORDS)