R-type Calcium Channels Research Articles

Dendritic NMDA Receptors Activate Axonal Calcium Channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.

Characterization of voltage operated R-type Ca2+ channels in modulating somatostatin receptor subtype 2- and 3-dependent inhibition of insulin secretion from INS-1 cells

Somatostatin (SST) inhibits Ca2+ entry into pancreatic B-cells via voltage-operated Ca2+ channels (VOCCs) of L-type, leading to the suppression of insulin secretion. Activation of R-type channels increases insulin secretion. However, the role of R-type Ca2+ channels (CaV2.3) in mediating the effects of SST on insulin secretion has not been so far investigated. Here, we identify the SST-receptor subtypes (SSTR) expressed on insulin-producing INS-1 cells by RT-PCR and by functional assays. The role of R-type channels in regulating [Ca2+]i in response to SST-treatment was detected by cell fluorescence imaging and patch-clamp technique. INS-1 expressed SSTR2 and SSTR3 and agonists (ag.) selective for these receptors reduced 10 nM exendin-4/20 mM glucose-stimulated insulin secretion. Surprisingly, SST and SST2-ag. transiently increased [Ca2+]i. Subsequently, these agonists led to a decrease in [Ca2+]i below the basal levels. In contrast, SST3-ag. failed to induce a transient peak of [Ca2+]i. Instead, a persistent minor suppression of [Ca2+]i was detected from 25 min. R-type channel blocker SNX-482 altered [Ca2+]i in SST- and SST2-ag.-treated cells. Notably, the inhibition of insulin secretion by SST and SST2-ag., but not SST3-ag. was attenuated by SNX-482. Taken together, SST and SSTR2 regulate [Ca2+]i and insulin secretion in INS-1 cells via R-type channels. In contrast, the R-type calcium channel does not mediate the effects of SST3-ag. on insulin secretion. We conclude that R-type channels play a major role in the inhibition of insulin secretion by somatostatin in INS-1 cells.

W1348 Function of R-Type Calcium Channels in Subsets of Myenteric Neurons in the Guinea-Pig Ileum

W1348 Function of R-Type Calcium Channels in Subsets of Myenteric Neurons in the Guinea-Pig Ileum

C101, a novel 4-amino-piperidine derivative selectively blocks N-type calcium channels

N-type Ca 2+ channels located on presynaptic nerve terminals regulate neurotransmitter release, including that from the spinal terminations of primary afferent nociceptors. Pharmacological and ion-channel gene knockdown approaches in animals have revealed N-type Ca 2+ channels to be particularly attractive molecular targets for the discovery and development of new analgesic drugs. In recent years, some non-peptide small molecular N-type Ca 2+ channel blockers have been reported. However, low selectivity and some side effects limit their further development. To overcome these disadvantages, some new compounds were designed and synthesized in our institute by optimizing the 4-amino-piperidine template. C101, one of these compounds, was demonstrated to block N-type Ca 2+ channels with higher selectivity. It was found that C101 produced concentration-dependent inhibition on N-type Ca 2+ channels expressed in Xenopus oocytes with an IC 50 is 2.2 ± 0.6 μM. The current–voltage relationship was not altered after 2-min exposure to C101. However, the steady-state inactivation relationship curve was shifted to more negative potentials for channels. Therefore, it seemed that C101 blocks the inactivated channel. C101 did not present any remarkable effects on voltage-gated potassium, sodium channels in cultured rat hippocampal neurons, and L-, P/Q-, R-type calcium channels and HERG channels expressed in Xenopus oocytes. The results suggested that C101 was a high selective blocker targeting N-type Ca 2+ channels, and may have a potential to be developed as a novel analgesic agent.

Effects of celecoxib on voltage-gated calcium channel currents in rat pheochromocytoma (PC12) cells

Cyclooxygenase-2 (COX-2) plays crucial roles in the development and invasion of tumors. Celecoxib, a selective COX-2 inhibitor, has been shown to be chemopreventive against cancer. However, to date, the mechanisms of these effects remain unclear. In this study, we investigate the effects of celecoxib on voltage-gated calcium channel (VGCC) currents in undifferentiated pheochromocytoma (PC12) cells using whole-cell patch clamp. Our results showed that celecoxib, instead of rofecoxib or NS-398, another selective COX-2 inhibitor, reversibly inhibited the current density of VGCC in a concentration-dependent manner, but had no apparent effects on the cells treated with nifedipine (1 μM), an L-type calcium channel blocker. Upon pre-incubation of PC12 cells with omega-conotoxia GVIA (1 μM), an N-type calcium channel blocker, omega-agatoxin IVA (1 μM), a P/Q-type calcium channel blocker, or SNX-482 (1 μM), a R-type calcium channel blocker, celecoxib (1 μM) inhibited the currents by 36%, 28%, and 25%, respectively. Celecoxib up-shifted the current–voltage ( I– V), and hyperpolarizedly shifted the inactivation curve, but did not markedly affect the activation curve. Intracellular application of H89, a protein kinase A inhibitor, failed to affect the celecoxib's VGCC currents inhibition. Taken together, our present results suggested that celecoxib inhibited L-type calcium channels in PC12 cells via a COX-2 independent pathway, which might be responsible for its clinical effects including anti-tumor.

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain.

Molecular Basis of Cav2.3 Calcium Channels in Rat Nociceptive Neurons

Ca(v)2.3 calcium channels play an important role in pain transmission in peripheral sensory neurons. Six Ca(v)2.3 isoforms resulting from different combinations of three inserts (inserts I and II in the II-III loop and insert III in the carboxyl-terminal region) have been identified in different mammalian tissues. To date, however, Ca(v)2.3 isoforms unique to primary sensory neurons have not been identified. In this study, we determined Ca(v)2.3 isoforms expressed in the rat trigeminal ganglion neurons. Whole tissue reverse transcription (RT)-PCR analyses revealed that only two isoforms, Ca(v)2.3a and Ca(v)2.3e, are present in TG neurons. Using single cell RT-PCR, we found that Ca(v)2.3e is the major isoform, whereas Ca(v)2.3e expression is highly restricted to small (<16 mum) isolectin B4-negative and tyrosine kinase A-positive neurons. Ca(v)2.3e was also preferentially detected in neurons expressing the nociceptive marker, transient receptor potential vanilloid 1. Single cell RT-PCR following calcium imaging and whole-cell patch clamp recordings provided evidence of an association between an R-type calcium channel component and Ca(v)2.3e expression. Our results suggest that Ca(v)2.3e in sensory neurons may be a potential target for the treatment of pain.

Modified sympathetic regulation in N-type calcium channel null-mouse

To elucidate the physiological importance of neuronal (N)-type calcium channels in sympathetic controls, we analyzed N-type channel-deficient (NKO) mice. Immunoprecipitation analysis revealed increased interaction between β3 (a major accessory subunit of N-type channels) and R-type channel-forming CaV2.3 in NKO mice. R–R intervals in NKO ECG recordings were elongated and fluctuating, suggesting disturbed sympathetic tonus. N-type channel inhibitors elongated the R–R interval in control mice, whereas R-type channel blocking with SNX-482 significantly affected NKO but not control mice, indicating a compensatory role for R-type channels. Echocardiography and Langendorff heart analysis confirmed a major role for R-type channels in NKO mice. Combined, our biochemical and physiological analyses strongly suggest that the remaining sympathetic tonus in NKO mice is dependent on R-type calcium channels.

Subunits of voltage-gated calcium channels in murine spiral ganglion cells

Conclusion. The results show that α1D, α1E, α2/δ, β1, and β3 subunits are expressed in spiral ganglion cells (SGCs), and the coexpression of α1D and α1E suggests the presence of L-type and R-type calcium channels in mammalian SGCs. Objective. To investigate the types of subunits of voltage-gated calcium channels in SGCs of the mouse. Materials and methods. SGCs were isolated from cochleae of neonatal mice and cultured for 24 h. Total RNA was extracted from cultured cells. After reverse transcription, the resulting cDNA was amplified by PCR with primers targeted to nucleotide sequences corresponding to seven different calcium channel subunits. The types of calcium channel subunits were identified by PCR analysis and nucleotide sequencing. Results. RT-PCR showed the strong and consistent amplification of α1D, α1E, α2/δ, β1, and β3 subunits from the mRNA of SGCs, and nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs.

Pharmacology and Drug Discovery for T-Type Calcium Channels

Voltage-gated calcium channels are found in the plasma membrane of many excitable and non-excitable cells. When open, they permit influx of calcium, which acts as a second messenger to initiate diverse physiological cellular processes. Ten unique alpha1 subunits, grouped in three families (CaV1, CaV2, and CaV3), encode biophysically and pharmacologically distinct low-voltage-activated T-type and high-voltage-activated L-type, N-type, P/Q-type, and R-type calcium channels. T-type calcium channels are found in neurons where they generate low-threshold calcium spikes and influence action potential firing patterns, in heart cells where they influence pacemaking and impulse conduction, in smooth muscle cells where they regulate myogenic tone and proliferation, in endocrine cells where they regulate hormone secretion, and in sperm where they regulate the acrosome reaction. Validation of T-type calcium channels in disease is based on an abundance of data pertaining to clinical efficacy of T-type calcium channel blockers in certain human conditions as well as information relating to the distribution, functional properties, and physiological roles of these channels. This review focuses on the cellular and molecular pharmacology of T-type calcium channels. It describes novel research approaches to discover potent and selective T-type calcium channel modulators as potential drugs for treating human disease and as tools for understanding better the physiological roles of T-type calcium channels.

Neurokinin 1 Receptors Trigger Overlapping Stimulation and Inhibition of CaV2.3 (R-Type) Calcium Channels

Neurokinin (NK) 1 receptors and CaV2.3 calcium channels are both expressed in nociceptive neurons, and mice lacking either protein display altered responses to noxious stimuli. Here, we examined modulation of CaV2.3 through NK1 receptors expressed in human embryonic kidney 293 cells. We find that NK1 receptors generate complex modulation of CaV2.3. In particular, weak activation of these receptors evokes mainly stimulation of CaV2.3, whereas strong receptor activation elicits profound inhibition that overlaps with channel stimulation. Unlike R-type channels encoded by CaV2.3, L-type (CaV1.3), N-type (CaV2.2), and P/Q-type (CaV2.1) channels are inhibited, but not stimulated, through NK1 receptors. Pharmacological experiments show that protein kinase C (PKC) mediates stimulation of CaV2.3 through NK1 receptors. The signaling mechanisms underlying inhibition were explored by expressing proteins that buffer either Galpha(q/11) (regulator of G protein signaling protein 3T and carboxyl-terminal region of phospholipase C-beta1) or Gbeta gamma subunits (transducin and the carboxyl-terminal region of bovine G-protein-coupled receptor kinase). A fast component of inhibition was attenuated by buffering Gbeta gamma, whereas a slow component of inhibition was reduced by buffering Galpha(q/11). When both Gbeta gamma and Galpha(q/11) were simultaneously buffered in the same cells, inhibition was virtually eliminated, but receptor activation still triggered substantial stimulation of CaV2.3. We also report that NK1 receptors accelerate the inactivation kinetics of CaV2.3 currents. Altogether, our results indicate that NK1 receptors modulate CaV2.3 using three different signaling mechanisms: a fast inhibition mediated by Gbeta gamma, a slow inhibition mediated by Galpha(q/11), and a slow stimulation mediated by PKC. This new information concerning R-type calcium channels and NK1 receptors may help in understanding nociception, synaptic plasticity, and other physiological processes.

An activity-dependent increased role for L-type calcium channels in exocytosis is regulated by adrenergic signaling in chromaffin cells

Chromaffin cells of the adrenal medulla represent a primary output of the sympathetic nervous system. Their electrical stimulation evokes the fusion of large dense core granules with the cell membrane and the exocytic release of multiple transmitter molecules into the circulation. There the transmitters contribute to the regulation of basic metabolism of the organism. Under physiological activity, granule fusion and transmitter release are limited by activity-dependent Ca2+ influx, entering through multiple isoforms of voltage-gated calcium channels. In this study we utilize perforated-patch voltage-clamp recordings and depolarize mouse chromaffin cells in situ with action potential-like waveforms to mimic physiological firing. We measure calcium influx through specific isoforms and measure cell capacitance as an index of granule fusion. Combining these approaches we calculate specific stimulus-secretion efficiencies for L-type, N-type, P/Q-type and R-type calcium channels under varied physiological activity levels. Current influx through all channel subtypes exhibited an activity-dependent depression. As expected P/Q-type channels, while responsible for modest Ca2+ influx, are tightly coupled to catecholamine secretion under all conditions. We further find that stimulation designed to match sympathetic input under the acute stress response recruits L-type channels to a state of enhanced stimulus-secretion efficiency. N- and R-type channels do not undergo activity-dependent recruitment and remain loosely coupled to the secretion. Thus, only L-type channels exhibit activity-dependent changes in their stimulus-secretion function under physiological stimulation. Lastly, we show that treatment with the β-adrenergic agonist, isoproterenol, specifically blocks the increase in the stimulus-secretion function of L-type channels. Thus, increased cell firing specifically enhances stimulus-secretion coupling of L-type Ca2+ channels in chromaffin cells in situ. This mechanism is regulated by an adrenergic signaling pathway.

Cocaine withdrawal enhances long‐term potentiation induced by corticotropin‐releasing factor at central amygdala glutamatergic synapses via CRF1, NMDA receptors and PKA

Cocaine addiction is an enduring, relapsing, behavioural disorder in which stressors reinstate cocaine-seeking even after prolonged abstinence. Evidence suggests that the 'anxiety-like' behaviour and stress associated with protracted withdrawal may be mediated by increased corticotropin-releasing factor (CRF) in the central nucleus of the amygdala (CeA), a part of the limbic circuitry engaged in the coding and transmission of stimulus-reward associations. In the present study we describe a long-lasting potentiation of glutamatergic transmission induced at lateral amygdala (LA)-to-CeA synapses by rat/human CRF. After 2 weeks of withdrawal from repeated intermittent exposure to cocaine, CRF-induced long-term potentiation (LTP) was greatly enhanced compared to the respective saline control group while, after short-term withdrawal (24 h), there was no significant difference between the two treatment groups, indicating alterations in CRF systems during protracted withdrawal from chronic cocaine. After prolonged withdrawal, CRF-induced LTP was dependent on activation of CRF2, CaV2.3 (R-type) calcium channels and intracellular signalling through protein kinase C in both saline- and cocaine-treated groups. The enhanced CRF-induced LTP after 2 weeks of withdrawal was mediated through augmented CRF1 receptor function, associated with an increased signalling through protein kinase A, and required N-methyl-D-aspartate (NMDA) receptors. Accordingly, single-cell recordings revealed a significantly increased NMDA/AMPA ratio after prolonged withdrawal from the cocaine treatment. These results support a role for CRF1 receptor antagonists as plausible treatment options during withdrawal from chronic cocaine and suggest Ca(V)2.3 blockers as potential candidates for pharmaceutical modulation of CRF systems.

Muscarinic modulation of Cav2.3 (R-type) calcium channels is antagonized by RGS3 and RGS3T

Ca(2+) influx through voltage-gated R-type (Ca(V)2.3) Ca(2+) channels is important for hormone and neurotransmitter secretion and other cellular events. Previous studies have shown that Ca(V)2.3 is both inhibited and stimulated through signaling mechanisms coupled to muscarinic ACh receptors. We previously demonstrated that muscarinic stimulation of Ca(V)2.3 is blocked by regulator of G protein signaling (RGS) 2. Here we investigated whether muscarinic inhibition of Ca(V)2.3 is antagonized by RGS3. RGS3 is particularly interesting because it contains a lengthy ( approximately 380 residue) amino-terminal domain of uncertain physiological function. Ca(V)2.3, M(2) muscarinic ACh receptors (M(2)R), and various deletion mutants of RGS3, including its native isoform RGS3T, were expressed in HEK293 cells, and agonist-dependent inhibition of Ca(V)2.3 was quantified using whole cell patch-clamp recordings. Full-length RGS3, RGS3T, and the core domain of RGS3 were equally effective in antagonizing inhibition of Ca(V)2.3 through M(2)R. These results identify RGS3 and RGS3T as potential physiological regulators of R-type Ca(2+) channels. Furthermore, they suggest that the signaling activity of RGS3 is unaffected by its extended amino-terminal domain. Confocal microscopy was used to examine the intracellular locations of four RGS3-enhanced green fluorescent protein fusion proteins. The RGS3 core domain was uniformly distributed throughout both cytoplasm and nucleus. By contrast, full-length RGS3, RGS3T, and the amino-terminal domain of RGS3 were restricted to the cytoplasm. These observations suggest that the amino terminus of RGS3 may serve to confine it to the cytoplasmic compartment where it can interact with cell surface receptors, heterotrimeric G proteins, and other signaling proteins.

Requirement of dendritic calcium spikes for induction of spike‐timing‐dependent synaptic plasticity

Spike-timing-dependent synaptic plasticity (STDP) by definition requires the temporal association of pre- and postsynaptic action potentials (APs). Yet, in cortical pyramidal neurons pairing unitary EPSPs with single APs at low frequencies is ineffective at generating plasticity. Using recordings from synaptically coupled layer 5 pyramidal neurons, we show here that high-frequency (200 Hz) postsynaptic AP bursts, rather than single APs, are required for both long-term potentiation (LTP) induction and NMDA channel activation during EPSP-AP pairing at low frequencies. Furthermore, we find that AP bursts can lead to LTP induction and NMDA channel activation during EPSP-AP pairing at both positive and negative times. High-frequency AP bursts generated supralinear calcium signals in basal dendrites suggesting the generation of dendritic calcium spikes, as has been observed previously in apical dendrites during AP burst firing at frequencies greater than 100 Hz. Consistent with a role of these dendritic calcium spikes in LTP induction, pairing EPSPs with low frequency (50 Hz) AP bursts was ineffective in generating LTP. Furthermore, supralinear calcium signals in basal dendrites during AP bursts were blocked by low concentrations of the T- and R-type calcium channel antagonist nickel, which also blocked LTP and NMDA channel activation. These data suggest an important role of dendritic calcium spikes during AP bursts in determining both the efficacy and time window for STDP induction.

Cannabinoids Modulate the P-Type High-Voltage-Activated Calcium Currents in Purkinje Neurons

Endocannabinoids released by postsynaptic cells inhibit neurotransmitter release in many central synapses by activating presynaptic cannabinoid CB1 receptors. In particular, in the cerebellum, endocannabinoids inhibit synaptic transmission at granule cell to Purkinje cell synapses by modulating presynaptic calcium influx via N-, P/Q-, and R-type calcium channels. Using whole cell patch-clamp techniques, we show that in addition to this presynaptic action, both synthetic and endogenous cannabinoids inhibit P-type calcium currents in isolated rat Purkinje neurons independent of CB1 receptor activation. The IC50 for the anandamide (AEA)-induced inhibition of P-current peak amplitude was 1.04 +/- 0.04 microM. In addition, we demonstrate that all the tested cannabinoids in a physiologically relevant range of concentrations strongly accelerate inactivation of P currents. The effects of AEA cannot be attributed to the metabolism of AEA because a nonhydrolyzing analogue of AEA, methanandamide inhibited P-type currents with a similar efficacy. All effects of cannabinoids on P-type Ca2+ currents were insensitive to antagonists of CB1 cannabinoid or vanilloid TRPV1 receptors. In cerebellar slices, WIN 55,212-2 significantly affected spontaneous firing of Purkinje neurons in the presence of CB1 receptor antagonist, in a manner similar to that of a specific P-type channel antagonist, indicating a possible functional implication of the direct effects of cannabinoids on P current. Taken together these findings demonstrate a functionally important direct action of cannabinoids on P-type calcium currents.

Transmitter modulation of spike‐evoked calcium transients in arousal related neurons: muscarinic inhibition of SNX‐482‐sensitive calcium influx

Nitric oxide synthase (NOS)-containing cholinergic neurons in the laterodorsal tegmentum (LDT) influence behavioral and motivational states through their projections to the thalamus, ventral tegmental area and a brainstem 'rapid eye movement (REM)-induction' site. Action potential-evoked intracellular calcium transients dampen excitability and stimulate NO production in these neurons. In this study, we investigated the action of several arousal-related neurotransmitters and the role of specific calcium channels in these LDT Ca(2+)-transients by simultaneous whole-cell recording and calcium imaging in mouse (P14-P30) brain slices. Carbachol, noradrenaline and adenosine inhibited spike-evoked Ca(2+)-transients, while histamine, t-ACPD, a metabotropic glutamate receptor agonist, and orexin-A did not. Carbachol inhibition was blocked by atropine, was insensitive to blockade of G-protein-coupled inward rectifier (GIRK) channels and was not inhibited by nifedipine, omega-conotoxin GVIA or omega-agatoxin IVA, which block L-, N- and P/Q-type calcium channels, respectively. In contrast, SNX-482 (100 nm), a selective antagonist of R-type calcium channels containing the alpha1E (Cav2.3) subunit, attenuated carbachol inhibition of the somatic spike-evoked calcium transient. To our knowledge, this is the first demonstration of muscarinic inhibition of native SNX-482-sensitive R-channels. Our findings indicate that muscarinic modulation of these channels plays an important role in the feedback control of cholinergic LDT neurons and that inhibition of spike-evoked Ca(2+)-transients is a common action of neurotransmitters that also activate GIRK channels in these neurons. Because spike-evoked calcium influx dampens excitability, our findings suggest that these 'inhibitory' transmitters could boost firing rate and enhance responsiveness to excitatory inputs during states of high firing, such as waking and REM sleep.

Genetic evidence in Caenorhabditis elegans implicates a syntaxin-1A-binding protein as critical for presynaptic volatile anesthetic action

Among the many cellular actions of volatile anesthetics (VAs), inhibition of neurotransmitter release is one of the least understood. The presynaptic release machinery has been proposed as a presynaptic VA target. We have found that the effects of clinical concentrations of VAs on locomotion in the nematode Caenorhabditis elegans are markedly reduced by mutation of the presynaptic SNARE protein syntaxin-1A. The syntaxin mutation results in a truncated syntaxin product along with reduced amounts of wild-type syntaxin. We hypothesize that the truncated mutant syntaxin functions to antagonize VA action on some unknown VA target, presumably by physically interacting with it. We have produced additional syntaxin truncation mutants, and their testing revealed that only the first two helical domains of the protein are necessary and sufficient for conferring VA resistance. This region of the protein is known to interact with only a few proteins, including N/P/Q/R-type calcium channels and UNC-13. Testing of a null mutant of the only known N/P/Q/R-type calcium channel showed that it was not VA resistant. These data implicate UNC-13 as the most likely mediator of VA sensitivity and could explain several puzzling observations about VA effects on vertebrate neurotransmitter release.

R-Type Calcium Channels Contribute to Afterdepolarization and Bursting in Hippocampal CA1 Pyramidal Neurons

Action potentials in pyramidal neurons are typically followed by an afterdepolarization (ADP), which in many cells contributes to intrinsic burst firing. Despite the ubiquity of this common excitable property, the responsible ion channels have not been identified. Using current-clamp recordings in hippocampal slices, we find that the ADP in CA1 pyramidal neurons is mediated by an Ni2+-sensitive calcium tail current. Voltage-clamp experiments indicate that the Ni2+-sensitive current has a pharmacological and biophysical profile consistent with R-type calcium channels. These channels are available at the resting potential, are activated by the action potential, and remain open long enough to drive the ADP. Because the ADP correlates directly with burst firing in CA1 neurons, R-type calcium channels are crucial to this important cellular behavior, which is known to encode hippocampal place fields and enhance synaptic plasticity.

Topiramate Inhibits the Initiation of Plateau Potentials in CA1 Neurons by Depressing R‐type Calcium Channels

Cholinergic-dependent plateau potentials (PPs) are intrinsically generated conductances that can elicit ictal-type seizure activity. The aim of this study was to investigate the actions of topiramate (TPM) on the generation of PPs. We used whole-cell patch-clamp recordings from CA1 pyramidal neurons in rat hippocampal slices to examine the effects of TPM on the PPs. In current-clamp mode, action potentials evoked PPs after cholinergic receptor stimulation. Therapeutically relevant concentrations of TPM (50 microM) depressed the PPs evoked by action potentials. Surprisingly, in voltage-clamp mode, we discovered that the cyclic nucleotide-gated (CNG) current that underlies PP generation (denoted as I(tail)) was not depressed. However, significantly longer depolarizing voltage steps were required to elicit I(tail). This suggested that the calcium entry trigger for evoking PPs was depressed by TPM and not I(tail) itself. TPM had no effect on calcium spikes in control conditions; however, TPM did reduce calcium spikes after cholinergic-receptor stimulation. We recently found that R-type calcium spikes are enhanced by cholinergic-receptor stimulation. Therefore we isolated R-type calcium spikes with a cocktail containing tetrodotoxin, omega-conotoxin MVIIC, omega-conotoxin-GVIA, omega-agatoxin IVA, and nifedipine. R-type calcium spikes were significantly depressed by TPM. We also examined the effects of TPM on recombinant Ca(V)2.3 calcium channels expressed in tsA-201 cells. TPM depressed currents mediated by Ca(V)2.3 subunits by a hyperpolarizing shift in steady-state inactivation. We have found that TPM reduces ictal-like activity in CA1 hippocampal neurons through a novel inhibitory action of R-type calcium channels.