Postnatal remodeling of neuronal connectivity shapes mature nervous systems.^1,2,3 The pruning of exuberant connections involves cell-autonomous and non-cell-autonomous mechanisms, such as neuronal activity. Indeed, experience-dependent competition sculpts various excitatory neuronal circuits.^4,5,6,7,8,9 Moreover, activity has been shown to regulate growth cone motility and the stability of neurites and synaptic connections.^{10,11,12,13,14} However, whether inhibitory activity influences the remodeling of neuronal connectivity or how activity influences remodeling in systems in which competition is not clearly apparent is not fully understood. Here, we use the Drosophila mushroom body (MB) as a model to examine the role of neuronal activity in the developmental axon pruning of γ-Kenyon cells. The MB is a neuronal structure in insects, implicated in associative learning and memory,^15,16 which receives mostly olfactory input from the antennal lobe.^17,18 The MB circuit includes intrinsic neurons, called Kenyon cells (KCs), which receive inhibitory input from the GABAergic anterior paired lateral (APL) neuron among other inputs. The γ-KCs undergo stereotypic, steroid-hormone-dependent remodeling^19,20 that involves the pruning of larval neurites followed by regrowth to form adult connections.²¹ We demonstrate that silencing neuronal activity is required for γ-KC pruning. Furthermore, we show that this is mechanistically achieved by cell-autonomous expression of the inward rectifying potassium channel 1 (irk1) combined with inhibition by APL neuron activity likely via GABA-B-R1 signaling. These results support the Hebbian-like rule "use it or lose it," where inhibition can destabilize connectivity and promote pruning while excitability stabilizes existing connections.

IB4-positive maxillary trigeminal ganglion (TG) neurons are a subtype of afferent neurons involving nociception in orofacial regions, and excitability of these neurons is associated with orofacial nociceptive sensitivity. TREK-2 channel is a member of two-pore domain potassium (K2P) channel family mediating leak K+ currents. It has been shown previously that TREK-2 channel activity can be enhanced following GABAB receptor activation, leading to a reduction of cortical neuron excitability. In the present study, we have characterized TREK-2 channel expression on maxillary TG neurons and investigated the effect of the GABAB agonist baclofen on electrophysiological properties of small-sized maxillary TG neurons of rats. We show with immunohistochemistry that TREK-2 channels are predominantly expressed in small-sized IB4-positive maxillary TG neurons. Patch-clamp recordings on neurons in ex vivo TG preparations show that baclofen hyperpolarizes resting membrane potentials, increases outward leak currents, and decreases input resistances in IB4-positive maxillary TG neurons. Moreover, baclofen significantly reduces action potential (AP) firing in IB4-positive maxillary TG neurons. In contrast, baclofen shows no significant effect on electrophysiological properties of small-sized nociceptive-like and non-nociceptive-like maxillary trigeminal neurons that are IB4-negatve. Our results suggest that TREK-2 channel activity can be enhanced by baclofen, leading to reduced excitability of IB4-positive maxillary TG neurons. This finding provides new insights into the role of TREK-2 and GABAB receptors in controlling nociceptive sensitivity in orofacial regions, which may have therapeutic implications.

Primary afferent fibers express extrasynaptic GABA_A and GABA_B receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.

γ-Aminobutyric acid B receptors (GABA_BRs) are broadly expressed throughout the central nervous system where they play an important role in regulating neuronal excitability and synaptic transmission. GABA_BRs are G protein-coupled receptors that mediate slow and sustained inhibitory actions via modulation of several downstream effector enzymes and ion channels. GABA_BRs are obligate heterodimers that associate with diverse arrays of proteins to form modular complexes that carry out distinct physiological functions. GABA_BR-dependent signaling is fine-tuned and regulated through a multitude of mechanisms that are relevant to physiological and pathophysiological states. This review summarizes the current knowledge on GABA_BR signal transduction and discusses key factors that influence the strength and sensitivity of GABA_BR-dependent signaling in neurons.

Classically, GABA_B receptors are thought to regulate neuronal excitability via G-protein-coupled inwardly rectifying potassium (GIRK) channels. Recent data, however, indicate that GABA_B receptors can also activate two-pore domain potassium channels. Here, we investigate which potassium channels are coupled to GABA_B receptors in rat neocortical layer 5 and hippocampal CA1 pyramidal neurons. Bath application of the non-specific GIRK channel blocker barium (200 μm) abolished outward currents evoked by GABA_B receptors in CA1 pyramidal, but only partially blocked GABA_B responses in layer 5 neurons. Layer 5 and CA1 pyramidal neurons also showed differential sensitivity to tertiapin-Q, a specific GIRK channel blocker. Tertiapin-Q partially blocked GABA_B responses in CA1 pyramidal neurons, but was ineffective in blocking GABA_B responses in neocortical layer 5 neurons. Consistent with the idea that GABA_B receptors are coupled to two-pore domain potassium channels, the non-specific blockers quinidine and bupivacaine partially blocked GABA_B responses in both layer 5 and CA1 neurons. Finally, we show that lowering external pH, as occurs in hypoxia, blocks the component of GABA_B responses mediated by two-pore domain potassium channels in neocortical layer 5 pyramidal neurons, while at the same time revealing a GIRK channel component. These data indicate that GABA_B receptors in neocortical layer 5 and hippocampal CA1 pyramidal neurons are coupled to different channels, with this coupling pH dependent on neocortical layer 5 pyramidal neurons. This pH dependency may act to maintain constant levels of GABA_B inhibition during hypoxia by enhancing GIRK channel function following a reduction in two-pore domain potassium channel activity.

Neuronal voltage-gated calcium channels have evolved as one of the most important players for calcium entry into presynaptic endings responsible for the release of neurotransmitters. In turn, and to fine-tune synaptic activity and neuronal communication, numerous neurotransmitters exert a potent negative feedback over the calcium signal provided by G protein-coupled receptors. This regulation pathway of physiologic importance is also extensively exploited for therapeutic purposes, for instance in the treatment of neuropathic pain by morphine and other μ-opioid receptor agonists. However, despite more than three decades of intensive research, important questions remain unsolved regarding the molecular and cellular mechanisms of direct G protein inhibition of voltage-gated calcium channels. In this study, we revisit this particular regulation and explore new considerations.

Neuronal voltage-gated N-type (Cav2.2) calcium channels are expressed throughout the nervous system and regulate neurotransmitter release and hence synaptic transmission. They are predominantly modulated via G protein-coupled receptor activated pathways, and the well characterized Gβγ subunits inhibit Cav2.2 currents. Analgesic α-conotoxin Vc1.1, a peptide from predatory marine cone snail venom, inhibits Cav2.2 channels by activating pertussis toxin-sensitive Gi/o proteins via the GABAB receptor (GABA(B)R) and potently suppresses pain in rat models. Using a heterologous GABA(B)R expression system, electrophysiology, and mutagenesis, we showed α-conotoxin Vc1.1 modulates Cav2.2 via a different pathway from that of the GABA(B)R agonists GABA and baclofen. In contrast to GABA and baclofen, Vc1.1 changes Cav2.2 channel kinetics by increasing the rate of activation and shifting its half-maximum inactivation to a more hyperpolarized potential. We then systematically truncated the GABA(B)(1a) C terminus and discovered that removing the proximal carboxyl terminus of the GABA(B)(1a) subunit significantly reduced Vc1.1 inhibition of Cav2.2 currents. We propose a novel mechanism by which Vc1.1 activates GABA(B)R and requires the GABA(B)(1a) proximal carboxyl terminus domain to inhibit Cav2.2 channels. These findings provide important insights into how GABA(B)Rs mediate Cav2.2 channel inhibition and alter nociceptive transmission.

N-type (Ca(v)2.2) voltage-gated calcium channels (VGCC) transduce electrical activity into other cellular functions, regulate calcium homeostasis and play a major role in processing pain information. Although the distribution and function of these channels vary widely among different classes of neurons, they are predominantly expressed in nerve terminals, where they control neurotransmitter release. To date, genetic and pharmacological studies have identified that high-threshold, N-type VGCCs are important for pain sensation in disease models. This suggests that N-type VGCC inhibitors or modulators could be developed into useful drugs to treat neuropathic pain. This review discusses the role of N-type (Ca(v)2.2) VGCCs in nociception and pain transmission through primary sensory dorsal root ganglion (DRG) neurons (nociceptors). It also outlines the potent and selective inhibition of N-type VGCCs by conotoxins, small disulfide-rich peptides isolated from the venom of marine cone snails. Of these conotoxins, ω-conotoxins are selective N-type VGCC antagonists that preferentially block nociception in inflammatory pain models, and allodynia and/or hyperalgesia in neuropathic pain models. Another conotoxin family, α-conotoxins, were initially proposed as competitive antagonists of muscle and neuronal nicotinic acetylcholine receptors (nAChR). Surprisingly, however, α-conotoxins Vc1.1 and RgIA, also potently inhibit N-type VGCC currents in the sensory DRG neurons of rodents and α9 nAChR knockout mice, via intracellular signaling mediated by G protein-coupled GABAB receptors. Understanding how conotoxins inhibit VGCCs is critical for developing these peptides into analgesics and may result in better pain management. This article is part of a Special Issue entitled: Calcium channels.

Underlying below-level cutaneous hypersensitivity observed following spinal cord injury (SCI) is a concurrent loss of inhibition with an increase in excitation in the spinal dorsal horn. Thus, a dual pharmacological approach, increasing spinal γ-aminobutyrate (GABA) inhibition and decreasing N-methyl-d-aspartate (NMDA) receptor-mediated excitation, could be more beneficial than either approach alone. The current study evaluated the antinociceptive effects of lumbar intrathecal (i.t.) administration of GABA receptor agonists and NMDA receptor antagonists alone and in combination in rats with neuropathic SCI pain. Rats developed markedly decreased hind paw withdrawal thresholds following an acute thoracic spinal cord compression, indicative of below-level hypersensitivity. Separately, i.t. GABA(A) receptor agonist muscimol and GABA(B) receptor agonist baclofen demonstrated dose-dependent antinociception, whereas i.t. NMDA receptor antagonist ketamine and the endogenous peptide [Ser¹]histogranin, a putative NMDA receptor antagonist, demonstrated no efficacy. The combination of baclofen and ketamine resulted in a supra-additive (synergistic) antinociception whereas the combinations with muscimol were merely additive. Intrathecal pretreatment with the GABA(B) receptor antagonist CGP 35348 prevented the antinociceptive effect of the baclofen and ketamine combination. The data indicate that blocking spinal NMDA receptors alone is not sufficient to ameliorate SCI hypersensitivity, whereas a combined approach, simultaneous activation of spinal GABA(B) receptors and NMDA receptor blockade with ketamine, leads to significant antinociception. By engaging diverse pain modulating systems at the spinal level, combination drug treatment may be a useful approach in treating neuropathic SCI pain.

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity. γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system, and mediates its effects via two classes of receptors: the GABA(A) and GABA(B) receptors. GABA(A) receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission. GABA(B) receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission. The extent of inhibitory neurotransmission is determined by a variety of factors, such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g., phosphorylation), as well as by the number of receptors present in the plasma membrane available for signal transduction. The level of GABA(B) receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation. In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA(B) receptors in the plasma membrane, and thereby signaling strength.

Despite evidence that high-affinity GABA(A) receptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low-affinity currents dominate those detected in acutely dissociated DRG neurons in vitro. This observation raises the possibility that high-affinity receptors are normally trafficked out of the DRG toward central and peripheral terminals. We therefore hypothesized that with time in culture, there would be an increase in high-affinity GABA(A) currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 h (acute) and 24 h (cultured) after plating with whole-cell patch-clamp techniques, Western blot, and semiquantitative reverse transcriptase polymerase chain reaction (sqRT-PCR) analysis. GABA(A) current density increases dramatically with time in culture in association with the emergence of two persistent currents with EC50's of 0.25±0.01 μM and 3.2±0.02 μM for GABA activation. In a subpopulation of neurons, there was also an increase in the potency of GABA activation of the transient current from an EC50 of 78.16±10.1 μM to 9.56±1.3 μM with time in culture. A fraction of the high-affinity current was potentiated by δ-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). δ-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute, but the membrane fraction in cultured, DRG neurons, with no detectable change in δ-subunit mRNA. However, the emergence of a high-affinity current blocked by THIP and insensitive to bicuculline was detected in a subpopulation of cultured neurons as well in association with an increase in ρ2- and ρ3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit-containing GABA(A) receptors are normally trafficked out of the DRG where they are targeted to peripheral and central processes. They also highlight that the interpretation of data obtained from cultured DRG neurons should be made with caution.

In the early neonatal period activation of GABAB receptors attenuates calcium current through N-type calcium channels while enhancing current through L-type calcium channels in rat hippocampal neurons. The attenuation of N-type calcium current has been previously demonstrated to occur through direct interactions of the βγ subunits of Gi/o G-proteins, but the signal transduction pathway for the enhancement of L-type calcium channels in mammalian neurons remains unknown. In the present study, calcium currents were elicited in acute cultures from postnatal day 6-8 rat hippocampi in the presence of various modulators of protein kinase A (PKA) and protein kinase C (PKC) pathways. Overnight treatment with an inhibitor of Gi/o (pertussis toxin, 200 ng/ml) abolished the attenuation of calcium current by the GABAB agonist, baclofen (10 μM) with no effect on the enhancement of calcium current. These data indicate that while the attenuation of N-type calcium current is mediated by the Gi/o subtype of G-protein, the enhancement of L-type calcium current requires activation of a different G-protein. The enhancement of the sustained component of calcium current by baclofen was blocked by PKC inhibitors, GF-109203X (500 nM), chelerythrine chloride (5 μM), and PKC fragment 19-36 (2 μM) and mimicked by the PKC activator phorbol-12-myristate-13-acetate (1 μM). The enhancement of the sustained component of calcium current was blocked by PKA inhibitors H-89 (1 μM) and PKA fragment 6-22 (500 nM) but not Rp-cAMPS (30 μM) and it was not mimicked by the PKA activator, 8-Br-cAMP (500 μM-1 mM). The data suggest that activation of PKC alone is sufficient to enhance L-type calcium current but that PKA may also be involved in the GABAB receptor mediated effect.

GABA(B) receptors have been found to play a key role in regulating membrane excitability and synaptic transmission in the brain. The GABA(B) receptor is a G-protein coupled receptor (GPCR) that associates with a subset of G-proteins (pertussis toxin sensitive Gi/o family), that in turn regulate specific ion channels and trigger cAMP cascades. In this review, we describe the relationships between the GABA(B) receptor, its effectors and associated proteins that mediate GABA(B) receptor function within the brain. We discuss a unique feature of the GABA(B) receptor, the requirement for heterodimerization to produce functional receptors, as well as an increasing body of evidence that suggests GABA(B) receptors comprise a macromolecular signaling heterocomplex, critical for efficient targeting and function of the receptors. Within this complex, GABA(B) receptors associate specifically with Gi/o G-proteins that regulate voltage-gated Ca(2+) (Ca(V)) channels, G-protein activated inwardly rectifying K(+) (GIRK) channels, and adenylyl cyclase. Numerous studies have revealed that lipid rafts, scaffold proteins, targeting motifs in the receptor, and regulators of G-protein signaling (RGS) proteins also contribute to the function of GABA(B) receptors and affect cellular processes such as receptor trafficking and activity-dependent desensitization. This complex regulation of GABA(B) receptors in the brain may provide opportunities for new ways to regulate GABA-dependent inhibition in normal and diseased states of the nervous system.

Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca(2+) channels (VGCCs) are especially important as Ca(2+) serves as both a charge carrier and second messenger. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, (Isl2b:EGFP)(ZC7), were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca(2+) current (HVA- and LVA-I(Ca), respectively). Ni(+)-sensitive LVA-I(Ca) occur in the minority of R-B neurons (30%) and ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels underlie the vast majority (90%) of HVA-I(Ca). To identify G protein coupled receptors (GPCRs) that modulate HVA-I(Ca), a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of I(Ca) appears to be transduced primarily via a cholera toxin-sensitive Gα subunit. These results provide the basis for using the zebrafish model system to understanding Ca(2+) channel function, and in turn, how Ca(2+) channels contribute to mechanosensory function.

Defined pharmacologically by its insensitivity to the GABA(A) antagonist bicuculline and sensitivity to the GABA analogue baclofen, the G protein-linked gamma-aminobutyric acid type B (GABA(B)) receptor couples to adenylyl cyclase, voltage-gated calcium channels, and inwardly-rectifying potassium channels. On the basis of a wealth of preclinical data in conjunction with early clinical observations that baclofen improves symptoms of gastroesophageal reflux disease (GERD), the GABA(B) receptor has been proposed as a therapeutic target for a number of diseases including GERD. Subsequently, there has been a significant effort to develop a peripherally-restricted GABA(B) agonist that is devoid of the central nervous system side effects that are observed with baclofen. In this article we review the in vitro and in vivo pharmacology of the peripherally-restricted GABA(B) receptor agonists and the preclinical and clinical development of lesogaberan (AZD3355, (R)-(3-amino-2-fluoropropyl) phosphinic acid), a potent and predominately peripherally-restricted GABA(B) receptor agonist with a preclinical therapeutic window superior to baclofen.

alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha9alpha10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the omega-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPbetaS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA(B) receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify Ca(V)2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABA(B) receptors.

The heterotrimeric G-protein-coupled receptors (GPCR) represent the largest and most diverse family of cell surface receptors and proteins. GPCR are widely distributed in the peripheral and central nervous systems and are one of the most important therapeutic targets in pain medicine. GPCR are present on the plasma membrane of neurons and their terminals along the nociceptive pathways and are closely associated with the modulation of pain transmission. GPCR that can produce analgesia upon activation include opioid, cannabinoid, alpha2-adrenergic, muscarinic acetylcholine, gamma-aminobutyric acidB (GABAB), groups II and III metabotropic glutamate, and somatostatin receptors. Recent studies have led to a better understanding of the role of these GPCR in the regulation of pain transmission. Here, we review the current knowledge about the cellular and molecular mechanisms that underlie the analgesic actions of GPCR agonists, with a focus on their effects on ion channels expressed on nociceptive sensory neurons and on synaptic transmission at the spinal cord level.

Developing networks follow common rules to shift from silent cells to coactive networks that operate via thousands of synapses. This review deals with some of these rules and in particular those concerning the crucial role of the neurotransmitter gamma-aminobuytric acid (GABA), which operates primarily via chloride-permeable GABA(A) receptor channels. In all developing animal species and brain structures investigated, neurons have a higher intracellular chloride concentration at an early stage leading to an efflux of chloride and excitatory actions of GABA in immature neurons. This triggers sodium spikes, activates voltage-gated calcium channels, and acts in synergy with NMDA channels by removing the voltage-dependent magnesium block. GABA signaling is also established before glutamatergic transmission, suggesting that GABA is the principal excitatory transmitter during early development. In fact, even before synapse formation, GABA signaling can modulate the cell cycle and migration. The consequence of these rules is that developing networks generate primitive patterns of network activity, notably the giant depolarizing potentials (GDPs), largely through the excitatory actions of GABA and its synergistic interactions with glutamate signaling. These early types of network activity are likely required for neurons to fire together and thus to "wire together" so that functional units within cortical networks are formed. In addition, depolarizing GABA has a strong impact on synaptic plasticity and pathological insults, notably seizures of the immature brain. In conclusion, it is suggested that an evolutionary preserved role for excitatory GABA in immature cells provides an important mechanism in the formation of synapses and activity in neuronal networks.

Little is known concerning coupling of cerebral GABA(B) receptors to G protein subtypes, and the influence of positive allosteric modulators (PAMs) has not been evaluated. These questions were addressed by an antibody-capture/scintillation proximity assay strategy. GABA concentration-dependently enhanced the magnitude of [(35)S]GTPgammaS binding to Galphao and, less markedly, Galphai(1/3) in cortex, whereas Gq and Gs/olf were unaffected. (R)-baclofen and SKF97581 likewise activated Galphao and Galphai(1/3), expressing their actions more potently than GABA. Similar findings were acquired in hippocampus and cerebellum, and the GABA(B) antagonist, CGP55845A, abolished agonist-induced activation of Galphao and Galphai(1/3) in all structures. The PAMs, GS39783, CGP7930 and CGP13501, inactive alone, enhanced efficacy and potency of agonist-induced [(35)S]GTPgammaS binding to Galphao in all regions, actions abolished by CGP55845A. In contrast, they did not modify efficacies at Galphai(1/3). Similarly, in human embryonic kidney cells expressing GABA(B(1a+2)) or GABA(B(1b+2)) receptors, allosteric modulators did not detectably enhance efficacy of GABA at Galphai(1/3), though they increased its potency. To summarise, GABA(B) receptors coupled both to Galphao and to Galphai, but not Gq and Gs/olf, in rat brain. PAMs more markedly enhanced efficacy of coupling to Go versus Gi(1/3). It will be of interest to confirm these observations employing complementary techniques and to evaluate their potential therapeutic significance.

Presynaptic gamma-aminobutyric acid type B receptors (GABA(B)Rs) regulate transmitter release at many central synapses by inhibiting Ca(2+) channels. However, the mechanisms by which GABA(B)Rs modulate neurotransmission at descending terminals synapsing on motoneurons in the spinal cord remain unexplored. To address this issue, we characterized the effects of baclofen, an agonist of GABA(B)Rs, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of the dorsolateral funiculus (DLF) terminals in a slice preparation from the turtle spinal cord. We found that baclofen depressed neurotransmission in a dose-dependent manner (IC(50) of approximately 2 microM). The membrane time constant of the motoneurons did not change, whereas the amplitude ratio of the evoked EPSPs in response to a paired pulse was altered in the presence of the drug, suggesting a presynaptic mechanism. Likewise, the use of N- and P/Q-type Ca(2+) channel antagonists (omega-conotoxin GVIA and omega-agatoxin IVA, respectively) also depressed EPSPs significantly. Therefore, these channels are likely involved in the Ca(2+) influx that triggers transmitter release from DLF terminals. To determine whether the N and P/Q channels were regulated by GABA(B)R activation, we analyzed the action of the toxins in the presence of baclofen. Interestingly, baclofen occluded omega-conotoxin GVIA action by approximately 50% without affecting omega-agatoxin IVA inhibition, indicating that the N-type channels are the target of GABA(B)Rs. Lastly, the mechanism underlying this effect was further assessed by inhibiting G-proteins with N-ethylmaleimide (NEM). Our data show that EPSP depression caused by baclofen was prevented by NEM, suggesting that GABA(B)Rs inhibit N-type channels via G-protein activation.

G protein-coupled inwardly rectifying potassium channels (GIRK) are important for neuronal signaling and membrane excitability. In the present study, we intend to find whether GIRK channels express functionally in adult rat dorsal root ganglion (DRG) neurons.

We used RT-PCR to detect mRNA for 4 subunits of GIRK in the adult DRG. The whole-cell patch clamp recording was used to confirm GIRK channels functionally expressed.

The mRNA for the 4 subunits of GIRK were detected in the adult DRG. GTPgammaS enhanced inwardly rectifying potassium (K+) currents of the DRG neurons, while Ba2+ inhibited such currents. Furthermore, the GIRK channels were shown to be coupled to the GABA(B) receptor, a member of the G protein-coupled receptor family, as baclofen increased the inwardly rectifying K+ currents.

GIRK channels are expressed and functionally coupled with GABA(B) receptors in adult rat DRG neurons.

GABA(B) receptors inhibit mechanosensitivity of visceral afferents. This is associated with reduced triggering of events that lead to gastro-esophageal reflux, with important therapeutic consequences. In other neuronal systems, GABA(B) receptor activation may be linked via G-proteins to reduced N-type Ca(2+) channel opening, increased inward rectifier K(+) channel opening, plus effects on a number of intracellular messengers. Here we aimed to determine the role of Ca(2+) and K(+) channels in the inhibition of vagal afferent mechanoreceptor function by the GABA(B) receptor agonist baclofen. The responses of three types of ferret gastro-esophageal vagal afferents (mucosal, tension and tension mucosal receptors) to graded mechanical stimuli were investigated in vitro. The effects of baclofen (200 microM) alone on these responses were quantified, and the effects of baclofen in the presence of the G-protein-coupled inward rectifier potassium channel blocker Rb(+) (4.7 mM) and/or the N-type calcium channel blocker omega-conotoxin GVIA (0.1 microM). Baclofen inhibition of mucosal receptor mechanosensitivity was abolished by both blockers. Its inhibitory effect on tension mucosal receptors was partly reduced by both. The inhibitory effect of baclofen on tension receptors was unaffected. The data indicate that the inhibitory action of GABA(B) receptors is mediated via different pathways in mucosal, tension and tension mucosal receptors via mechanisms involving both N-type Ca(2+) channels and inwardly rectifying K(+) channels and others.

Biochemical and structural studies of signaling proteins have revealed critical features of peptide motifs at the interaction surfaces between proteins. Such information can be used to design small peptides that can be used as functional probes of specific interactions in signaling cascades. This article describes the use of a novel domain (the GoLoco motif) found in several members of the regulators of G-protein signaling (RGS) protein family to probe the specificity of Galpha subunit involvement in the coupling of dopamine and somatostatin receptors to ion channels in the AtT20 neuroendocrine cell line. Peptides encoding the GoLoco motifs of RGS12 and AGS3 were perfused into single cells during electrical recording of agonist-induced current responses by whole cell patch clamp methods. The particular sequences chosen have been demonstrated to bind selectively to the GDP-bound form of Galphai, but not Galphao, and preclude association of Gbetagamma and Galphai subunits. A functional manifestation of this property is observed in the progressive uncoupling of D2 dopamine receptors and Kir3.1/3.2 channels with repeated agonist application. Similar uncoupling is not observed with somatostatin receptors nor with D2 receptors coupling to calcium channels, suggesting Galpha subunit specificity in these signaling pathways. Motifs found in other proteins in the GPCR signaling machinery may also prove useful in assessing G-protein signaling specificity and complexity in single cells in the future.

Our goal was to test the following hypotheses: 1) GABA(A) receptors facilitate neurokinin release from primary afferent terminals; 2) they do this by suppressing an inhibitory effect of GABA(B) receptors; 3) the activation of these two receptors is controlled by the firing frequency of primary afferents. We evoked neurokinin release by stimulating the dorsal root attached to spinal cord slices, and measured it using neurokinin 1 receptor (NK1R) internalization. Internalization evoked by root stimulation at 1 Hz (but not at 100 Hz) was increased by the GABA(A) receptor agonists muscimol (effective concentration of drug for 50% of the increase [EC50] 3 microM) and isoguvacine (EC50 4.5 microM). Internalization evoked by root stimulation at 100 Hz was inhibited by the GABA(A) receptor antagonists bicuculline (effective concentration of drug for 50% of the inhibition [IC50] 2 microM) and picrotoxin (IC50 243 nM). Internalization evoked by incubating the root with capsaicin (to selectively recruit nociceptive fibers) was increased by isoguvacine and abolished by picrotoxin. Therefore, GABA(A) receptors facilitate neurokinin release. Isoguvacine-facilitated neurokinin release was inhibited by picrotoxin, low Cl-, low Ca2+, Ca2+ channel blockers and N-methyl-D-aspartate receptor antagonists. Bumetanide, an inhibitor of the Na(+)-K(+)-2Cl- cotransporter, inhibited isoguvacine-facilitated neurokinin release, but this could be attributed to a direct inhibition of GABA(A) receptors. The GABA(B) agonist baclofen inhibited NK1R internalization evoked by 100 Hz root stimulation (IC50 1.5 microM), whereas the GABA(B) receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP-55845) increased NK1R internalization evoked by 1 Hz root stimulation (EC50 21 nM). Importantly, baclofen inhibited isoguvacine-facilitated neurokinin release, and CGP-55845 reversed the inhibition of neurokinin release by bicuculline. In conclusion, 1) GABA(B) receptors located presynaptically in primary afferent terminals inhibit neurokinin release; 2) GABA(A) receptors located in GABAergic interneurons facilitate neurokinin release by suppressing GABA release onto these GABA(B) receptors; 3) high frequency firing of C-fibers stimulates neurokinin release by activating GABA(A) receptors and inhibiting GABA(B) receptors, whereas low frequency firing inhibits neurokinin release by the converse mechanisms.

There are many different calcium channels expressed in the mammalian nervous system, but N-type and P/Q-type calcium channels appear to dominate the presynaptic terminals of central and peripheral neurons. The neurotransmitter-induced modulation of these channels can result in alteration of synaptic transmission. This review highlights the mechanisms by which neurotransmitters affect the activity of N-type and P/Q-type calcium channels. The inhibition of these channels by voltage-dependent and voltage-independent mechanisms is emphasized because of the wealth of information available on the intracellular mediators and on the effect of these pathways on the single-channel gating.

GABA(B) receptors are broadly expressed in the nervous system and have been implicated in a wide variety of neurological and psychiatric disorders. The cloning of the first GABA(B) receptor cDNAs in 1997 revived interest in these receptors and their potential as therapeutic targets. With the availability of molecular tools, rapid progress was made in our understanding of the GABA(B) system. This led to the surprising discovery that GABA(B) receptors need to assemble from distinct subunits to function and provided exciting new insights into the structure of G protein-coupled receptors (GPCRs) in general. As a consequence of this discovery, it is now widely accepted that GPCRs can exist as heterodimers. The cloning of GABA(B) receptors allowed some important questions in the field to be answered. It is now clear that molecular studies do not support the existence of pharmacologically distinct GABA(B) receptors, as predicted by work on native receptors. Advances were also made in clarifying the relationship between GABA(B) receptors and the receptors for gamma-hydroxybutyrate, an emerging drug of abuse. There are now the first indications linking GABA(B) receptor polymorphisms to epilepsy. Significantly, the cloning of GABA(B) receptors enabled identification of the first allosteric GABA(B) receptor compounds, which is expected to broaden the spectrum of therapeutic applications. Here we review current concepts on the molecular composition and function of GABA(B) receptors and discuss ongoing drug-discovery efforts.

Nerve growth factor (NGF) promotes the survival of embryonic sensory neurons and maintains the phenotypic characteristics of primary nociceptive neurons postnatally. NGF also contributes to nociceptor activation and hyperalgesia during inflammatory pain states. The purpose of this study was to determine whether NGF might have an additional pronociceptive action by interfering with opioid-mediated analgesia in primary nociceptive neurons. Sensory neurons were isolated from the dorsal root ganglia of weanling rats and kept in standard culture conditions either with or without exogenous NGF (50 ng/ml). Currents through voltage-gated calcium channels were recorded from individual neurons using the whole cell patch clamp technique with Ba(2+) as the charge carrier (I(Ba)). The micro-opioid agonist fentanyl (1 microM) and the GABA(B) agonist baclofen (50 microM) were used to test G protein-dependent inhibition of I(Ba). Fentanyl inhibited I(Ba) by an average of 38+/-4% in untreated cells vs. 25+/-2% in NGF-treated cells (P<0.01). NGF had no effect on I(Ba) current magnitude or kinetics. The NGF-induced attenuation of opioid action was observed as early as 4 h after exposure, but was not seen when NGF was applied by bath perfusion for up to 40 min, suggesting that the effect was not mediated by a rapid phosphorylation event. The effect of NGF was prevented by K-252a (100 nM), an inhibitor of TrkA autophosphorylation. Baclofen-induced inhibition of I(Ba), on the other hand, was not affected by NGF treatment, suggesting that NGF modulation of opioid-mediated inhibition occurred upstream from the G protein. This was supported by the finding that GTP-gamma-S, an agonist independent G protein activator, inhibited I(Ba) similarly in both untreated and NGF treated cells. The results show that NGF selectively attenuated opioid-mediated inhibition of I(Ba) via TrkA receptor activation, possibly by altering opioid receptor function.

Calcium influx into any cell requires fine tuning to guarantee the correct balance between activation of calcium-dependent processes, such as muscle contraction and neurotransmitter release, and calcium-induced cell damage. G protein-coupled receptors play a critical role in negative feedback to modulate the activity of the CaV2 subfamily of the voltage-dependent calcium channels, which are largely situated on neuronal and neuro-endocrine cells. The basis for the specificity of the relationships among membrane receptors, G proteins, and effector calcium channels will be discussed, as well as the mechanism by which G protein-mediated inhibition is thought to occur. The inhibition requires free G beta gamma dimers, and the cytoplasmic linker between domains I and II of the CaV2 alpha 1 subunits binds G beta gamma dimers, whereas the intracellular N terminus of CaV2 alpha 1 subunits provides essential determinants for G protein modulation. Evidence suggests a key role for the beta subunits of calcium channels in the process of G protein modulation, and the role of a class of proteins termed "regulators of G protein signaling" will also be described.

Capsaicin stimulates neurokinin release in the spinal cord when applied both centrally and peripherally. To determine whether these two actions have different mechanisms, we measured neurokinin 1 receptor (NK1R) internalization in rat spinal cord slices elicited by incubating the whole slice or just the dorsal root with capsaicin. NK1R internalization produced by incubating the slices with capsaicin was abolished by the NK1R antagonist RP-67580, by the vanilloid receptor 1 (VR1) antagonist capsazepine, and by eliminating Ca(2+) from the medium, but was not affected by the Na(+) channel blocker lidocaine. Therefore, the internalization was due to neurokinin release mediated by Ca(2+) entry through VR1 receptors, but did not require the firing of action potentials. Incubating the root with capsaicin produced NK1R internalization in the ipsilateral dorsal horn that was abolished when capsazepine or lidocaine was included in, or when Ca(2+) was omitted from, the medium surrounding the root. Therefore, the internalization was mediated by Ca(2+) entry in the axons through VR1, and required firing of action potentials. The efficacy of capsaicin when applied to the root (36+/-3%) was lower than when applied to the slice (91+/-3%), but its potency was the same (0.49 microM and 0.37 microM, respectively). We also investigated whether presynaptic N-methyl-D-aspartate (NMDA) and GABA(B) receptors modulate these two actions of capsaicin. Neither the NMDA receptor blocker MK-801 nor the GABA(B) agonist baclofen decreased NK1R internalization produced by 1 microM capsaicin applied to the slices, but they inhibited the internalization produced by 0.3 microM capsaicin applied to the slices or 1 microM capsaicin applied to the root. Therefore, capsaicin can produce neurokinin release from primary afferents 1) by a direct action on their central terminals and 2) by increasing the firing of action potentials on their axons. The first effect largely bypasses other modulatory mechanism, but the second does not.

The effects of the GABAB-selective agonist baclofen were studied on guinea pig nodose ganglion neurones using grease gap and intracellular recording techniques, and on peripheral mechanosensitive endings in the guinea pig oesophagus and stomach with extracellular recordings. GABA dose-dependently reduced the amplitude of the compound action potential of C-type neurones (C spikes, EC50 = 30.9 microM), which was prevented by the GABAA antagonist bicuculline (10 microM). The GABAB agonist baclofen (1-300 microM) did not produce any significant effect on the amplitude of C spikes. In microelectrode studies, baclofen (100 microM) evoked hyperpolarisation (by 2.53 +/- 0.51 mV, n = 6, N = 5) in a subset of nodose neurones (6 out of 26, N = 18). In seven out of eight neurones (N = 8) with a slow after-hyperpolarisation following action potentials, baclofen significantly inhibited its amplitude by 19 +/- 4% (n = 7, p < 0.05). GABA (100 microM) evoked a depolarisation of 9.3 +/- 2.4 mV (10 nodose neurones, N = 9, p < 0.05) associated with a decrease in input impedance of 49 +/- 12% (N = 4, p < 0.05). Baclofen (100-200 microM) did not affect either spontaneous or stretch-evoked firing of distension-sensitive vagal mechanoreceptors of the guinea pig oesophagus and stomach but did inhibit mechanoreceptors in the ferret oesophagus. Antibodies to GABAB receptor 1a splice variants labelled most of the neurones and numerous fibres in the guinea pig nodose ganglion while antibodies to GABAB receptor 1b splice variants stained only nerve cell bodies. There were numerous nerve fibres showing GABAB receptor 1a- and 1b-like immunoreactivity in the myenteric plexus in the guinea pig oesophagus and stomach but not in anterogradely labelled extrinsic vagal nerve fibres. The result indicates that most guinea pig C-type nodose ganglion neurones have GABAB receptors on their cell bodies but their density on distension-sensitive peripheral endings is too low to allow modulation of mechanotransduction. There is a significant species-dependent difference in the expression of GABAB receptors on peripheral vagal mechanosensitive endings.

Efficient transfection of NG108-15 cells with GABA(B) receptor subunits was achieved using polyethylenimine. Baclofen modulated high voltage-activated Ca(2+) current in differentiated cells transfected with GABA(B1) and GABA(B2) receptor subunits or with the GABA(B2) subunit alone, but not with the GABA(B1) subunit alone. Characteristics of the current modulation were very similar for cells transfected with GABA(B1/2) and GABA(B2) subunits. Using antisense oligonucleotides against GABA(B1) subunits and also western immunoblotting, we are able to show that NG108-15 cells contain endogenous GABA(B1) subunits. Therefore, functional receptors can be formed by the combination of native GABA(B1) subunits with transfected GABA(B2) subunits, in agreement with the proposed heteromeric structure of GABA(B) receptors. Finally, we used selective channel blockers to identify the subtypes of Ca(2+) channels that are modulated by GABA(B) receptors. In fact, in differentiated NG108-15 cells, the recombinant GABA(B) receptors couple only to N-type Ca(2+) channels.

Functional human GABA(B(1a,2)) and GABA(B(1b,2)) receptors have been stably expressed in mammalian CHO K1 cells. Detailed characterization of GABA(B) ligand binding at each of the receptors has been compared using [(3)H]-CGP54626A. In cell membranes fractions, [(3)H]-CGP54626A bound to a single site with a K(D) of 1. 51+/-1.12 nM, B(max) of 2.02+/-0.17 pmoles mg protein(-1) and 0. 86+/-0.20 nM, B(max) of 5.19+/-0.57 pmoles mg protein(-1) for GABA(B(1a,2)) and GABA(B(1b,2)) respectively. In competition binding assays the rank order was identical for both GABA(B) receptors. For known GABA(B) agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABA(B) antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 > or =2-OH Saclofen > or =ABPA. The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl(2) from the binding assay conditions was ligand dependent to either cause a decrease in ligand affinity or to have no significant effect. However, these effects were similar for both GABA(B) receptors. A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABA(B) receptors. In competition assays, the rank order was the same for both GABA(B(1a,2)) and GABA(B(1b,2)) and consistent with that seen with cell membrane fractions. These data suggest that, in terms of ligand binding, the currently identified isoforms of the GABA(B) receptor are pharmacologically indistinguishable.

Activation of gamma-aminobutyric acid(B) (GABA(B)) receptors in dorsal root ganglion (DRG) neurones leads to inhibition of calcium (Ca(2+)) channels. The role of the GABA(B1) receptor subunit was assessed by its depletion achieved by microinjection of DRG neurones with an antisense (A/S) oligodeoxynucleotide (ODN). Control neurones were injected with a scrambled version of the A/S ODN (missense) or were not injected. Patch clamp recordings of Ca(2+) channel current were made two to four days after injection. GABA(B1) A/S substantially reduced the current inhibition induced by baclofen, a GABA(B) agonist. Therefore, most, if not all, native GABA(B) receptors which couple to Ca(2+) channels contain GABA(B1). Moreover, if native receptors are heterodimers of GABA(B1) and GABA(B2), then GABA(B2), in isolation, is unable to sustain coupling to Ca(2+) channels.

Neuronal GABA(B) receptors regulate calcium and potassium currents via G-protein-coupled mechanisms and play a critical role in long-term inhibition of synaptic transmission in the CNS. Recent studies have demonstrated that assembly of GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits into functional heterodimers is required for coupling to potassium channels in heterologous systems. However whether heterodimerization is required for the coupling of GABA(B) receptors to effector systems in neurons remains to be established. To address this issue, we have studied the coupling of recombinant GABA(B) receptors to endogenous Ca(2+) channels in superior cervical ganglion (SCG) neurons using nuclear microinjection to introduce both sense and antisense expression constructs. Patch-clamp recording from neurons injected with both GABA(B)R1a/1b and GABA(B)R2 cDNAs or with GABA(B)R2 alone produced marked baclofen-mediated inhibition of Ca(2+) channel currents via a pertussis toxin-sensitive mechanism. The actions of baclofen were blocked by CGP62349, a specific GABA(B) antagonist, and were voltage dependent. Interestingly, SCGs were found to express abundantly GABA(B)R1 but not GABA(B)R2 at the protein level. To determine whether heterodimerization of GABA(B)R1 and GABA(B)R2 subunits was required for Ca(2+) inhibition, the GABA(B)R2 expression construct was microinjected with a GABA(B)R1 antisense construct. This resulted in a dramatic decrease in the levels of the endogenous GABA(B)R1 protein and a marked reduction in the inhibitory effects of baclofen on Ca(2+) currents. Therefore our results suggest that in neurons heteromeric assemblies of GABA(B)R1 and GABA(B)R2 are essential to mediate GABAergic inhibition of Ca(2+) channel currents.

To examine the role of G(o) in modulation of ion channels by neurotransmitter receptors, we characterized modulation of ionic currents in hippocampal CA3 neurons from mice lacking both isoforms of Galpha(o). In CA3 neurons from Galpha(o)(-/-) mice, 2-chloro-adenosine and the GABA(B)-receptor agonist baclofen activated inwardly rectifying K(+) currents and inhibited voltage-dependent Ca(2+) currents just as effectively as in Galpha(o)(+/+) littermates. However, the kinetics of transmitter action were dramatically altered in Galpha(o)(-/-) mice in that recovery on washout of agonist was much slower. For example, recovery from 2-chloro-adenosine inhibition of calcium current was more than fourfold slower in neurons from Galpha(o)(-/-) mice [time constant of 12.0 +/- 0.8 (SE) s] than in neurons from Galpha(o)(+/+) mice (time constant of 2.6 +/- 0.2 s). Recovery from baclofen effects was affected similarly. In neurons from control mice, effects of both baclofen and 2-chloro-adenosine on Ca(2+) currents and K(+) currents were abolished by brief exposure to external N-ethyl-maleimide (NEM). In neurons lacking Galpha(o), some inhibition of Ca(2+) currents by baclofen remained after NEM treatment, whereas baclofen activation of K(+) currents and both effects of 2-chloro-adenosine were abolished. These results show that modulation of Ca(2+) and K(+) currents by G protein-coupled receptors in hippocampal neurons does not have an absolute requirement for Galpha(o). However, modulation is changed in the absence of Galpha(o) in having much slower recovery kinetics. A likely possibility is that the very abundant Galpha(o) is normally used but, when absent, can readily be replaced by G proteins with different properties.

Whole-cell patch-clamp recordings were obtained from nodose ganglion neurons acutely dissociated from 10-30-day-old rats to characterize the Ca2+ channel types that are modulated by GABA(B) and mu-opioid receptors. Five components of high-threshold current were distinguished on the basis of their sensitivity to blockade by omega-conotoxin GVIA, nifedipine, omega-agatoxin IVA and omega-conotoxin MVIIC. Administration of the mu-opioid agonist H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol (0.3-1 mM) or the GABA(B) agonist baclofen in saturating concentrations suppressed high-threshold Ca2+ currents by 49.9+/-2.4% (n=69) and 18.7+/-2.1% (n=35), respectively. The inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol exceeded that by baclofen in virtually all neurons that responded to both agonists (67%), and occlusion experiments revealed that responses to mu-opioid and GABA(B) receptor activation were not linearly additive. In addition, administration of staurosporine, a non-selective inhibitor of protein kinase A and C, did not affect the inhibitory responses to either agonist or prevent the occlusion of baclofen-induced current inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol. Blockade of N-type channels by omega-conotoxin GVIA eliminated current suppression by baclofen in all cells tested (n=11). Mu-opioid-induced inhibition in current was abolished by omega-conotoxin GVIA in 12 of 30 neurons tested, but was only partially reduced in the remaining 18 neurons. In the latter cells administration of omega-agatoxin IVA reduced, but did not eliminate the mu-opioid sensitive current component that persisted after blockade of N-type channels. This residual component of mu-opioid-sensitive current was blocked completely by omega-conotoxin MVIIC in nine neurons, whereas responses to H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol were still recorded in the remaining cells after administration of these Ca2+ channel toxins and nifedipine. Dihydropyridine-sensitive (L-type) current was not affected by activation of mu-opioid or GABA(B) receptors in any of the neurons. These data indicate that in nodose ganglion neurons mu-opioid receptors are negatively coupled to N-, P- and Q-type channels as well as to a fourth, unidentified toxin-resistant Ca2+ channel. In contrast, GABA(B) receptors are coupled only to N-type channels. Furthermore, the results do not support a role for either protein kinase C or A in the modulatory pathway(s) coupling mu-opioid and GABA(B) receptors to Ca2+ channels, but rather lend credence to the notion that the signalling mechanisms utilized by these two receptors might simply compete for inhibitory control of a common pool of N-type channels.

Proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM) are novel hypotensive peptides. Although they are derived from the same gene product, proadrenomedullin, their hypotensive mechanisms are different; PAMP inhibits the release of norepinephrine from the peripheral sympathetic nerve endings, whereas AM fosters vasodilation by elevating intracellular cAMP, possibly via activation of cholera toxin-sensitive G proteins. In PC12 cells, PAMP inhibited N-type calcium channel via activation of pertussis toxin-sensitive mechanisms. To clarify the relationship between the hypotensive effect of PAMP and pertussis toxin-sensitive mechanisms, we administered pertussis vaccine intraperitoneally into rats for 3 consecutive days. By using mesenteric artery preparation, we showed that PAMP's ability to decrease norepinephrine overflow was significantly attenuated in pertussis toxin-treated rat (-18.5 +/- 6.9%; P<.05 versus control rats). In electrically stimulated pithed rat, PAMP (20 and 40 nmol/kg) showed a hypotensive effect (-13 +/- 5 and -18 +/- 7 mm Hg, respectively; P<.05, P<.01), whereas in pertussis vaccine-treated rat it did not (-2 +/- 3 and -8 +/- 9 mm Hg, respectively; P=NS). Also, in pithed rat, plasma norepinephrine level was significantly elevated by electrical stimulation in both control (0.323 +/- 0.035 ng/mL) and pertussis vaccine-treated groups (0.355 +/- 0.079 ng/mL). After injection of PAMP (40 nmol/kg), plasma norepinephrine level significantly decreased in the control group (0.225 +/- 0.044 ng/mL; P<.01) but not in the pertussis vaccine-treated group (0.392 +/- 0.021 ng/mL; P=NS). Moreover, in conscious rats, intravenous administration of PAMP (40 nmol/kg) did not evoke hypotension after pertussis vaccine treatment, although untreated controls had significantly decreased arterial pressure (-5 +/- 2 versus -20 +/- 3 mm Hg; P<.01). In contrast to PAMP, the administration of AM (1 nmol/kg) significantly reduced the blood pressure of pertussis vaccine-treated as well as control rats (-20 +/- 5 versus -18 +/- 7 mm Hg; P=NS). These results demonstrate that the ability of PAMP to inhibit norepinephrine release from peripheral sympathetic nerve endings and to decrease blood pressure is pertussis toxin sensitive. Our findings thus suggest that despite being derived from the same gene, PAMP and AM apparently produce hypotension by activating different signaling pathways.

Calcium enters the cytoplasm mainly via voltage-activated calcium channels (VACC), and this represents a key step in the regulation of a variety of cellular processes. Advances in the fields of molecular biology, pharmacology and electrophysiology have led to the identification of several types of VACC (referred to as T-, N-, L-, P/Q- and R-types). In addition to possessing distinctive structural and functional characteristics, many of these types of calcium channels exhibit differential sensitivities to pharmacological agents. In recent years a large number of toxins, mainly small peptides, have been purified from the venom of predatory marine cone snails and spiders. Many of these toxins have specific actions on ion channels and neurotransmitter receptors, and the toxins have been used as powerful tools in neuroscience research. Some of them (omega-conotoxins, omega-agatoxins) specifically recognize and block certain types of VACC. They have common structural backbones and some been synthesized with identical potency as the natural ones. Natural, synthetic and labeled calcium channel toxins have contributed to the understanding of the diversity of the neuronal calcium channels and their function. In particular, the toxins have been useful in the study of the role of different types of calcium channels on the process of neurotransmitter release. Neuronal calcium channel toxins may develop into powerful tools for diagnosis and treatment of neurological diseases.

Effects of riluzole on high voltage-activated (HVA) calcium channels of rat dorsal root ganglion neurons were studied using the whole-cell patch-clamp technique. Riluzole at 30 microM inhibited the HVA currents. The onset and offset of riluzole inhibitory effect were slow usually taking more than 3 min. Riluzole inhibition of the HVA currents was abolished and partially reduced by addition of 500 microM GDP-beta-S and 1 mM N-ethylmaleimide, respectively, to the pipette solution. Pre-treatment with pertussis toxin or application of depolarizing pre-pulses did not affect riluzole's inhibitory effect on the HVA currents. Riluzole inhibition of the HVA currents was also blocked by internal application of 50 microg/ml protein kinase A inhibitory peptide. It was concluded that pertussis toxin-insensitive G-proteins and protein kinase A may be involved in riluzole inhibition of the HVA currents.

The two isoforms of the dopamine D2 receptor, the D2short and the D2long differ in a 29 amino acid insert in the third cytoplasmic loop with which G proteins interact. We have previously reported that in rat melanotropes, expression of D2short increases markedly at the end of the first postnatal week which is concurrent with innervation of the intermediate lobe. Using immunohistochemistry, this study examined expression of G alpha i1/2, G alpha i3, G alpha o and G alpha s proteins before and after dopaminergic innervation. G alpha i3 increased through gestational day 20, and then remained level to postnatal day 6. At this time, coinciding with the induction of D2short expression, G alpha i3 immunoreactive intensity increased markedly, possibly indicating co-regulation of these proteins. On postnatal day 6, G alpha s immunoreactive intensity increased in some, but not all, melanotropes. The resulting heterogeneity in Gs expression persisted in the adult. G alpha i1/2 immunoreactivity did not change and G alpha o was detected only subsequent to the event of innervation. Thus, dopamine released from axons and acting through D2 receptor stimulation could increase G alpha i3 immunoreactivity and decrease G alpha s immunoreactive intensity in some melanotropes.