Kathleen Dunlap

Exocytotic Ca2+ channels in mammalian central neurons

Intracellular Ca2+ initiates physiological events as diverse as gene transcription, muscle contraction, cell division and exocytosis. Predictably, the metabolic machinery that elicits and responds to changes in intracellular Ca2+ is correspondingly heterogeneous. This review focuses on one element of this complex web that is of particular importance to neurobiologists: identifying which members of the voltage-dependent Ca(2+)-channel superfamily are responsible for the Ca2+ that enters nerve terminals and elicits vesicular release of chemical transmitters.

Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus

N-type calcium channels play a dominant role in controlling synaptic transmission in many peripheral neurons. Transmitter release from mammalian central nerve terminals, however, is relatively resistant to the N channel antagonist omega-conotoxin GVIA. We studied the sensitivity of glutamatergic synaptic transmission in rat hippocampal slices to omega-conotoxin and to omega-Aga-IVA, a P channel antagonist. Both toxins reduced the amplitude of excitatory postsynaptic potentials in CA1 pyramidal neurons, but omega-Aga-IVA was the more rapid and efficacious. These results were corroborated by biochemical studies measuring subsecond, calcium-dependent [3H]glutamate release from hippocampal synaptosomes. Thus, at least two calcium channel types trigger glutamate release from hippocampal neurons, but P-type plays a more prominent role. Eliminating synaptic transmission in the CNS, therefore, may require inhibiting more than a single calcium channel type.

Neurotransmitters decrease the calcium component of sensory neurone action potentials

RELEASE of neurotransmitters from presynaptic axon terminals requires the influx of Ca2+ ions during the nerve terminal action potential1. Action potentials recorded in some neurone cell bodies exhibit a relatively large Ca2+ component, and it has been suggested that these soma Ca2+ spikes may provide a model for Ca2+ influx across the less accessible nerve terminal membrane2. Recent data support the usefulness of this model. Serotonin (5-hydroxytryptamine, 5-HT) increases transmitter output at certain habituated sensory nerve-motoneurone synapses in the abdominal ganglion of Aplysia and it also prolongs the Ca2+ spike recorded in the sensory neurone cell body3. Enkephalin reduces the stimulated release of substance P by adult cat trigeminal neurones4 and by cultured embryonic chick dorsal root ganglion (DRG) neurones5, and it decreases the quantal content of excitatory postsynaptic potentials (e.p.s.ps, transmitter unknown) evoked in cultured rat spinal cord neurones by co-cultured DRG cells6. This peptide also decreases the duration and magnitude of the Ca2+ component of the DRG soma spike5. With the thought that modulation of Ca2+ currents may be a general correlate of presynaptic inhibition, we have studied the effect of several putative neurotransmitters on the soma spike of cultured chick sensory neurones, and report here that they decrease the calcium component of cell body action potentials.

Localization of calcium channels in Paramecium caudatum.

1. Electrical recordings from Paramecium caudatum were made after removal of the cilia with chloral hydrate and during ciliary regrowth to study the electrical properties of that portion of the surface membrane enclosing the ciliary axoneme. 2. Removal of the somatic cilia (a 50% reduction in membrane surface area) results in an almost complete elimination of the regenerative Ca response, all‐or‐none Ba2+ spike, and delayed rectification. 3. A twofold increase in input resistance resulted from the 50% reduction in membrane surface area. 4. The electrical properties remained unchanged, despite prolonged exposure to the chloral hydrate, until the cilia were mechanically removed. 5. Restoration of the Ca response accompanied ciliary regrowth, so that complete excitability returns when the cilia regain their original lengths. 6. It is concluded that the voltage‐sensitive Ca channels are localized to that portion of surface membrane surrounding the cilia. 7. Measurements of membrane constants before and after deciliation and estimations of the cable constants of a single cilium suggest that the cilia of Paramecium may be fully isopotential along their length and with the major cell compartment.

TWO TYPES OF γ‐AMINOBUTYRIC ACID RECEPTOR ON EMBRYONIC SENSORY NEURONES

1 Embryonic sensory neurones of the chick grown in dissociated cell culture respond to application of low concentrations of γ‐aminobutyric acid (GABA) with a change in resting membrane resistance (Rin) and/or a change in action potential duration (APD) (Dunlap & Fischbach, 1978; Choi & Fischbach, 1981). Intracellular microelectrode recording techniques were employed to determine if these two effects are mediated by the same, or different, GABA receptors. 2 Cells responded, for the most part, with a change in either Rin or APD, but 10% of the cells exhibited both effects. In the latter cells the two responses were clearly distinguishable as discussed below. 3 The proportion of neurones exhibiting a GABA‐induced decrease in Rin declined during the first week in vitro while the proportion exhibiting a decrease in APD increased during that time. 4 The two effects were pharmacologically distinct. Muscimol, a GABA analogue, produced only the change in Rin (ED50 = 5.5 μm) while baclofen, another analogue of GABA, produced only the change in APD (ED50 = 1 μm). The analogues were approximately equipotent with GABA. Bicuculline, a GABA antagonist, blocked the muscimol‐induced change in Rin (but not the baclofen‐induced change in APD) in a dose‐dependent fashion with an ID50 = 0.7 μm. 5 The time courses of the two effects were different. The change in APD resulting from a brief application of GABA (or baclofen) was prolonged relative to the rapid return to control associated with the GABA‐ (or muscimol‐) induced change in Rin. 6 Desensitization of the two responses exhibited separate time courses. In the continual presence of the agonists, GABA‐ and muscimol‐induced decreases in Rin completely desensitized in ca. 10 s while GABA‐ and baclofen‐induced decreases in APD persisted undiminished throughout a prolonged (1 min) application of the drugs and returned to control only after cessation of application. 7 It is concluded that embryonic chick sensory neurones in culture exhibit two types of GABA receptor that differ in their functional and pharmacological properties. Implications of these results are discussed.

Dihydropyridine inhibition of neuronal calcium current and substance P release

Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking45Ca influx and Ca-dependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons. Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cells normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.

G proteins as regulators of ion channel function

Virtually unknown just a decade ago, GTP-binding proteins (G proteins) have become a major focus of current research. This family of closely related proteins transduce extracellular signals (such as hormones, neurotransmitters and sensory stimuli) into effector responses (1,2). It is now evident that ion channel permeability is one such effector response. In fact, the striking increase in the frequency of reports that demonstrate G protein-regulated ion channel function suggests that channels whose permeability mechanism can be altered by a G protein-mediated process may be more the rule than the exception. It is well-known that the cAMP-dependent modulation of ion channels is under the control of G proteins that regulate adenylate cyclase activity(3,4). However recent studies demonstrate that G proteins also transduce agonist-induced changes in channel activity that do not involve adenylate cyclase. It is on this aspect of G protein signal transduction that this review will focus.

Transmitter-mediated inhibition of N-type calcium channels in sensory neurons involves multiple GTP-binding proteins and subunits

The modulation of voltage-activated Ca2+ channels by neurotransmitters and peptides is very likely a primary means of regulating Ca(2+)-dependent physiological functions such as neurosecretion, muscle contraction, and membrane excitability. In neurons, N-type Ca2+ channels (defined as omega-conotoxin GVIA-sensitive) are one prominent target for transmitter-mediated inhibition. This inhibition is widely thought to result from a shift in the voltage independence of channel gating. Recently, however, voltage-independent inhibition has also been described for N channels. As embryonic chick dorsal root ganglion neurons express both of these biophysically distinct modulatory pathways, we have utilized these cells to test the hypothesis that the voltage-dependent and -independent actions of transmitters are mediated by separate biochemical pathways. We have confirmed this hypothesis by demonstrating that the two modulatory mechanisms activated by a single transmitter involve not only different classes of G protein but also different G protein subunits.

Pharmacological characterization of presynaptic calcium channels using subsecond biochemical measurements of synaptosomal neurosecretion

The recent development of peptide antagonists that selectively block subtypes of neuronal calcium channel has provided tools to study the role of presynaptic calcium channels in triggering exocytosis. A variety of methods have consistently demonstrated that multiple channel types participate in exocytosis. We have studied the subsecond kinetics of [3H]glutamate release from rat cortical synaptosomes as an assay for presynaptic calcium channel activity. The system has been characterized over a broad range of conditions in an effort to compare biochemical measurements of transmitter release with electrophysiological measurements of synaptic currents. The efficacies of omega-agatoxin IVA and omega-conotoxins GVIA and MVIIC were increased when Ca2+ influx was decreased by: (1) decreasing the KCl concentration to diminish the extent of depolarization, (2) decreasing the Ca2+ concentration, or (3) partially blocking Ca2+ influx with one of the other antagonists. By using these toxins in combination, we found that at least three types of pharmacologically distinct channel participate in exocytosis. The largest fraction of glutamate release is blocked by omega-agatoxin IVA (IC50 = 12.2 nM) and by omega-conotoxin MVIIC (IC50 = 35 nM), consistent with the pharmacology of a P type channel. The effects of saturating concentrations (1 microM) of omega-agatoxin IVA or omega-conotoxin MVIIC occlude each other, suggesting that these peptides overlap completely. The specific N type antagonist omega-conotoxin GVIA inhibits a significant portion of release (IC50 less than 1 nM) but only under conditions of reduced Ca2+ concentration. These results suggest that the N type channel in nerve terminals is distinct from that found in hippocampal somata, since it appears to be resistant to by omega-conotoxin MVIIC. The combination of omega-conotoxin GVIA (100 nM) and either omega-agatoxin IVA or omega-conotoxin MVIIC (1 microM each) blocked approx 90% of release when the Ca2+ concentration was reduced (0.46 mM or less), but 30-40% of release remained when the concentration of Ca2+ in the stimulus buffer was 1 mM or greater, indicating that a resistant channel type(s) also participates in exocytosis. Specific inhibitors of this resistant phenotype will be useful for further refinement of our understanding of the role of presynaptic calcium channels in mediating neurosecretion.

CLASS A CALCIUM CHANNEL VARIANTS IN PANCREATIC ISLETS AND THEIR ROLE IN INSULIN SECRETION

The initiation of insulin release from rat islet β cells relies, in large part, on calcium influx through dihydropyridine-sensitive (α1D) voltage-gated calcium channels. Components of calcium-dependent insulin secretion and whole cell calcium current, however, are resistant toL-type channel blockade, as well as to ω-conotoxin GVIA, a potent inhibitor of α1B channels, suggesting the expression of additional exocytotic calcium channels in the islet. We used a reverse transcription-polymerase chain reaction-based strategy to ascertain at the molecular level whether the α1Acalcium channel isoform was also present. Results revealed two new variants of the rat brain α1A channel in the islet with divergence in a putative extracellular domain and in the carboxyl terminus. Using antibodies and cRNA probes specific for α1A channels, we found that the majority of cells in rat pancreatic islets were labeled, indicating expression of the α1A channels in β cells, the predominant islet cell type. Electrophysiologic recording from isolated islet cells demonstrated that the dihydropyridine-resistant current was sensitive to the α1A channel blocker, ω-agatoxin IVA. This toxin also inhibited the dihydropyridine-resistant component of glucose-stimulated insulin secretion, suggesting functional overlap among calcium channel classes. These findings confirm the presence of multiple high voltage-activated calcium channels in the rat islet and implicate a physiologic role for α1A channels in excitation-secretion coupling in β cells.