Presynaptic calcium stores and synaptic transmission

Following the gradual recognition of the importance of intracellular calcium stores for somatodendritic signaling in the mammalian brain, recent reports have also indicated a significant role of presynaptic calcium stores. Ryanodine-sensitive stores generate local, random calcium signals that shape spontaneous transmitter release. They amplify spike-driven calcium signals in presynaptic terminals, and consequently enhance the efficacy of transmitter release. They appear to be recruited by an association with certain types of calcium-permeant ion channels, and they induce specific forms of synaptic plasticity. Recent research also indicates a role of inositoltrisphosphate-sensitive presynaptic calcium stores in synaptic plasticity.     

D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.     

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive Gαi/o coupled receptors, which results in voltage-dependent inhibition of channel activity via Gβγ subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both Gβγ (i.e. voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

Presynaptic calcium channels: Pharmacology and regulation

Voltage-dependent Ca²⁺ channels are considered as molecular trigger elements for signal transmission at chemical synapses. Due to their central role in this fundamental process, function and pharmacology of presynaptic Ca²⁺ channels have recently been the subject of extensive exploration employing various experimental techniques. Several lines of evidence indicate that, at nerve terminals in higher vertebrates, the evoked influx of Ca²⁺-ions is mainly mediated by Ca²⁺ channels of the P-type. The stringent regulation of presynaptic Ca²⁺ channels is supposed to be involved in fine-tuning the efficiency of synaptic transmission. Intrinsic control mechanisms, such as voltage- or Ca²⁺-dependent inactivation, or modulation of channel activity, either by G-proteins directly or via phosphorylation by protein kinases, may be of particular functional importance.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive G alpha(i/o) coupled receptors, which results in voltage-dependent inhibition of channel activity via G betagamma subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both G betagamma (i.e., voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

Alternative splicing matters: N-type calcium channels in nociceptors

How many different calcium channels does it take to make a nervous system? The answer: more than any of us predicted. In 1975 Hagiwara and colleagues published the first evidence that functionally different calcium channels are expressed in cells. By 1999, the calcium channel family could boast ten members, each member defined by a unique set of attributes to support their cellular functions and by unique amino acid sequences. Although nine of these genes are expressed in the nervous system, that number still seemed insufficient to support the wide spectrum of neuronal functions controlled by voltage-gated calcium channels. This discrepancy is probably explained by alternative pre-messenger RNA splicing which substantially expands the number of protein activities available from a limited number of genes. Like many other ion channel genes, each Ca(V)alpha(1) gene has the capacity to generate perhaps thousands of unique splice isoforms with unique functional properties. The high level of conservation among alternatively spliced exons in Ca(V)2.2 genes of different species and in some cases closely related genes implies biological importance. A number of Ca(V)alpha(1) isoforms have been identified from neural tissue but until recently we lacked direct evidence linking a specific splice site in a calcium channel gene to a specific function in an identified neuron population. Our recent studies show that alternative pre-mRNA splicing of a pair of 32 amino acid encoding exons in the C-terminus of Ca(V)2.2, e37a and e37b, underlie the expression of two mutually exclusive N-type channel isoforms. The inclusion of e37a creates a module that couples the N-type channel to a powerful form of G protein-dependent inhibition. The inhibitory pathway that works through e37a is voltage-independent, requires G(i/o) and tyrosine kinase activation, and is used by mu opioid and GABA(B) receptors to downregulate N-type channel activity. Combined with our previous studies that show enrichment of e37a in nociceptors, our data suggest a molecular basis for the high susceptibility of N-type currents in sensory neurons to voltage-independent inhibition following G protein activation.     

Models of ion pores in N-type voltage-gated calcium channels

Two computer models of the outer vestibule of the pore of the N-type voltage-gated Ca²⁺ channel are predicted. The models are constructed from β-hairpin peptide segments in the S5–S6 loops of each of the four domains that produce the channel. These hairpins together are modeled to form a short eight-stranded β barrel. The models contain a ring of glutamates at the base of the barrel, which have been shown by mutagenesis experiments to function as a selectivity filter. These filters are suggested by the models to be of the correct dimensions to allow the permeation of a hydrated calcium ion, where the filter glutamates may substitute for molecules of water from the hydration shell of the ion. The models also suggest that a ring of threonines and an aspartate might be present between the mouth of the pore and the filter, and hence the models may prove useful in suggesting future mutagenesis experiments.     

Structure-activity relationships of omega-conotoxins at N-type voltage-sensitive calcium channels

Due to their selectivity towards voltage-sensitive calcium channels (VSCCs) omega-conotoxins are being exploited as a new class of therapeutics in pain management and may also have potential application in ischaemic brain injury. Here, the structure-activity relationships (SARs) of several omega-conotoxins including GVIA, MVIIA, CVID and MVIIC are explored. In addition, the three-dimensional structures of these omega-conotoxins and some structurally related peptides that form the cysteine knot are compared, and the effects of the solution environment on structure discussed. The diversity of binding and functional assays used to measure omega-conotoxin potencies at the N-type VSCC warranted a re-evaluation of the relationship between these assays. With one exception, [A22]-GVIA, this analysis revealed a linear correlation between functional (peripheral N-type VSCCs) and radioligand binding assays (central N-type VSCCs) for the omega-conotoxins and analogues that were tested over three studies. The binding and functional results of several studies are compared in an attempt to identify and distinguish those residues that are important in omega-conotoxin function as opposed to those that form part of the structural scaffold. Further to determining what omega-conotoxin residues are important for VSCC binding, the range of possible interactions between the ligand and channel are considered and the factors that influence the selectivity of MVIIA, GVIA and CVID towards N-type VSCCs examined.     

N型钙通道与疼痛

N型电压依赖性钙通道(VDCCs)在疼痛的传递与调控中具有重要作用。它们密集分布于脊髓背角伤害感受性神经元突触前末梢,参与主要疼痛介质如谷氨酸和P物质等释放的调节。通过阻断上述通道,选择性N型VDCCs阻断剂表现出强效镇痛作用,N型VDCCs Cav2．2亚基基因敲除小鼠也表现为痛阈提高。N型VDCCs还分布于自主神经系统和中枢神经系统突触部位,现有的N型VDCCs阻断剂用于疼痛治疗时出现的各种副作用与这些部位的突触抑制有关。最近发现,背根节伤害感受性神经元上存在一种特异的N型VDCCs亚型,这为疼痛治疗提供了一个非常有意义的新靶标。     

T-type calcium channels in synaptic plasticity

The role of T-type calcium currents is rarely considered in the extensive literature covering the mechanisms of long-term synaptic plasticity. This situation reflects the lack of suitable T-type channel antagonists that till recently has hampered investigations of the functional roles of these channels. However, with the development of new pharmacological and genetic tools, a clear involvement of T-type channels in synaptic plasticity is starting to emerge. Here, we review a number of studies showing that T-type channels participate to numerous homo- and hetero-synaptic plasticity mechanisms that involve different molecular partners and both pre- and post-synaptic modifications. The existence of T-channel dependent and independent plasticity at the same synapse strongly suggests a subcellular localization of these channels and their partners that allows specific interactions. Moreover, we illustrate the functional importance of T-channel dependent synaptic plasticity in neocortex and thalamus.     

Presynaptic calcium diffusion from various arrays of single channels. Implications for transmitter release and synaptic facilitation.

A one-dimensional model of presynaptic calcium diffusion away from the membrane, with cytoplasmic binding, extrusion by a surface pump, and influx during action potentials, can account for the rapid decay of phasic transmitter release and the slower decay of synaptic facilitation following one spike, as well as the very slow decline in total free calcium observed experimentally. However, simulations using this model, and alternative versions in which calcium uptake into organelles and saturable binding are included, fail to preserve phasic transmitter release to spikes in a long tetanus. A three-dimensional diffusion model was developed, in which calcium enters through discrete membrane channels and acts to release transmitter within 50 nm of entry points. Analytic solutions of the equations of this model, in which calcium channels were distributed in active zone patches based on ultrastructural observations, were successful in predicting synaptic facilitation, phasic release to tetanic spikes, and the accumulation of total free calcium. The effects of varying calcium buffering, pump rate, and channel number and distribution were explored. Versions appropriate to squid giant synapses and frog neuromuscular junctions were simulated. Limitations of key assumptions, particularly rapid nonsaturable binding, are discussed.     

Molecular determinants of Rem2 regulation of N-type calcium channels

Rem2 belongs to the RGK family of small GTPases whose members are known to interact with the voltage gated calcium channel β subunit, and to inhibit or abolish calcium currents. To identify the underlying functional domains of Rem2, we created several N- or C-terminally truncated Rem2 proteins and examined their abilities to interact with the Ca_v β subunit and to regulate the activities of Ca_v2.2 N-type calcium channels. Confocal imaging of Rem2 in tsA-201 cells revealed that it contains a membrane-targeting signal in its C-terminus, consistent with previous studies. Co-precipitation assays showed that Ca_v β₃ interaction depends on Rem2 residues 1-123. Only Rem2 proteins that targeted the cell membrane as well as bound the β subunit were able to reduce whole cell calcium currents.     

Molecular determinants of syntaxin 1 modulation of N-type calcium channels

We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939-2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain.     

Interactions among toxins that inhibit N-type and P-type calcium channels

A number of peptide toxins from venoms of spiders and cone snails are high affinity ligands for voltage-gated calcium channels and are useful tools for studying calcium channel function and structure. Using whole-cell recordings from rat sympathetic ganglion and cerebellar Purkinje neurons, we studied toxins that target neuronal N-type (Ca(V)2.2) and P-type (Ca(V)2.1) calcium channels. We asked whether different toxins targeting the same channels bind to the same or different sites on the channel. Five toxins (omega-conotoxin-GVIA, omega-conotoxin MVIIC, omega-agatoxin-IIIA, omega-grammotoxin-SIA, and omega-agatoxin-IVA) were applied in pairwise combinations to either N- or P-type channels. Differences in the characteristics of inhibition, including voltage dependence, reversal kinetics, and fractional inhibition of current, were used to detect additive or mutually occlusive effects of toxins. Results suggest at least two distinct toxin binding sites on the N-type channel and three on the P-type channel. On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance. On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers. For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.     

Presynaptic receptors for FMRFamide, histamine and buccalin regulate acetylcholine release at a neuro-neuronal synapse of Aplysia by modulating N-type Ca2+ channels.

At an identified neuro-neuronal synapse of the buccal ganglion of Aplysia, quantal release of acetylcholine (ACh) is increased by FMRFamide and decreased by histamine or buccalin. Activation of presynaptic receptors for these neuromodulators modifies a presynaptic Ca2+ current which is nifedipine-resistant and omega-conotoxin-sensitive. The voltage-sensitivity of these N-type Ca2+ channels is increased by FMRFamide and decreased by histamine through the intermediate of G proteins. Buccalin does not implicate G proteins and reduces the Ca2+ current without affecting the voltage-sensitivity of N-type Ca2+ channels. The possibility of relating the shifts in voltage-dependence of the Ca2+ current induced by FMRFamide and histamine to the phosphorylation state of the N-type Ca2+ channels is discussed. A scheme for the complex regulation of ACh release by presynaptic auto- and heteroreceptors is proposed.     

Structure-activity relationship study of 1,4-dihydropyridine derivatives blocking N-type calcium channels

Cilnidipine is a 1,4-dihydropyridine derived L/N-type calcium channel dual blocker possessing neuroprotective and analgesic effects which are related to its N-type calcium channel inhibitory activity. In order to find specific N-type calcium channel blockers with the least effects on cardiovascular system, we performed structure-activity relationship study on APJ2708, which is a derivative of cilnidipine, and found a promising N-type calcium channel blocker 21b possessing analgesic effect in vivo with a 1600-fold lower activity against L-type calcium channels than that of cilnidipine.     

Elementary events underlying voltage-dependent G-protein inhibition of N-type calcium channels.

Voltage-dependent G-protein inhibition of N-type calcium channels reduces presynaptic calcium entry, sharply attenuating neurotransmitter release. Studies in neurons demonstrate that G-proteins have multiple modulatory effects on N-type channels. The observed changes may reflect genuine complexity in G-protein action and/or the intricate interactions of multiple channels and receptors in neurons. Expression of recombinant M2-muscarinic receptors and N-type channels in HEK 293 cells allowed voltage-dependent inhibition to be studied in isolation. In this system, receptor-activated G-proteins had only one effect: a 10-fold increase in the time required for channels to first open following membrane depolarization. There were no changes in gating after the channel first opened, and unitary currents were not detectably altered by modulation. Despite its simplicity, this single change successfully accounts for the complex alterations in whole-cell current observed during G-protein inhibition in neurons.     

Cysteine string protein regulates G protein modulation of N-type calcium channels

Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a "J domain" and a palmitoylated cysteine-rich "string" region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. G. subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gbetagamma subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.     

Cysteine strings,calcium channels and synaptic transmission

Multidisciplinary studies have led to the discovery and characterization of cysteine string proteins (csps) in both Drosophila and Torpedo. Phenotypic analysis of csp mutants in Drosophila demonstrates a crucial role for csp in synaptic transmission. Expression studies of Torpedo csp (Tcsp) in Xenopus oocytes suggests that the protein has some role in the function of presynaptic Ca²⁺ channels. However, biochemical purification of Tcsp indicates that is associated with synaptic vesicles rather than with the plasma membrane of presynaptic terminals where Ca²⁺ channels reside. These results suggest a model in which csps serve as a link by which docked synaptic vesicles could modulate the activity of presynaptic Ca²⁺ channels.