Rat group I metabotropic glutamate receptors inhibit neuronal Ca2+ channels via multiple signal transduction pathways in HEK 293 cells

We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.     

Inhibition of N-type calcium channels: the only mechanism by which presynaptic alpha 2-autoreceptors control sympathetic transmitter release

Alpha 2-Adrenoceptors are known to inhibit voltage-dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic alpha 2-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10 microM nicotine, or by adding Ca2+ to otherwise Ca(2+)-free medium when cells had been made permeable by the calcium ionophore A23187 or by alpha-latrotoxin. Pretreatment of neurons with the N-type Ca2+ channel blocker omega-conotoxin GVIA and application of the alpha 2-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with alpha-latrotoxin or A23187. In contrast, the L-type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or alpha-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In omega-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with omega-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented alpha 2-adrenergic effects. Our results indicate that the alpha 2-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of omega-conotoxin GVIA-sensitive N-type Ca2+ channels. Mechanisms downstream of these channels and voltage-sensitive Ca2+ channels other than N-type appear not to be important.     

Differential regulation of N- and Q-type Ca2+ channels by cyclic nucleotides and G-proteins

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway delimited to the membrane. In addition, modulation of Ca2+ channel activity by protein kinases also contributes to the dynamic regulation of neuronal physiology. Recently, differences in these modulations between Ca2+ channel subtypes have been shown in several neuronal preparations. Here we show that two types of presynaptic Ca2+ channel (N-type and Q-type) are differentially regulated by cAMP and G-proteins using a Xenopus oocyte expression system. Treatment to increase cytosolic cAMP concentration with forskolin and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type channel current, and the enhancement was reversed by protein kinase A inhibitors. Much smaller enhancement was observed in N-type channel current after the cAMP elevation. When large depolarizing prepulse was applied to the oocytes for evaluation of the tonic inhibition of Ca2+ channels by intrinsic G-protein activity, N-type channel current elicited a large prepulse facilitation but Q-type channels did not. The tonic inhibition of N-type channels was abolished by an intracellular perfusion with a 'cut-open' recording configuration, or by co-expression with G(alpha o). When kappa opioid receptors were co-expressed and stimulated with agonists, depolarization-resistant inhibition was more apparent in Q-type channels than in N-type channels. These results suggest that Q-type channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels, and that activity of N-type channels can be more highly regulated in a voltage-dependent manner by G(betagamma) than that of Q-type channels. These differences may account for the selective regulation of neurotransmitter release by these Ca2+ channels.     

Activation of alpha 2-adrenoceptors causes inhibition of calcium channels but does not modulate inwardly-rectifying K+ channels in caudal raphe neurons

Many neurotransmitter receptors that interact with pertussis toxin-sensitive G proteins, including the alpha 2-adrenergic receptor, can modulate both voltage-dependent calcium channels and G protein-coupled inwardly-rectifying K+ channels. Serotonergic neurons of the medulla oblongata (nucleus raphe obscurus and nucleus raphe pallidus), which provide a major projection to sympathetic and motor output systems, receive a catecholaminergic input and express alpha 2-adrenergic receptors. Therefore, we tested the effects of norepinephrine on voltage-dependent calcium channels and G protein-coupled inwardly-rectifying K+ channels in neonatal raphe neurons using whole-cell recording in a brainstem slice preparation. Calcium channel currents were inhibited by norepinephrine in the majority of raphe neurons tested (88%) and in all identified tryptophan hydroxylase-immunoreactive (i.e. serotonergic) neurons. When tested in the same neurons, the magnitude of calcium current inhibition by norepinephrine (approximately 25%) was less than that induced by 5-hydroxytryptamine (approximately 50%). The norepinephrine-induced calcium current inhibition was mediated by alpha 2-adrenergic receptors; it was mimicked by UK 14304, an alpha 2-adrenergic receptor agonist and blocked by idazoxan, an alpha 2-adrenergic receptor antagonist, but not affected by prazosin or propanolol (alpha 1 and beta adrenergic antagonists, respectively). Calcium current inhibition by norepinephrine was essentially eliminated following application of omega-Conotoxin GVIA and omega-Agatoxin IVA, indicating that norepinephrine modulated N- and P/Q-type calcium channels predominantly. Calcium current inhibition by norepinephrine was voltage-dependent and mediated by pertussis toxin-sensitive G proteins. Thus, as expected, alpha 2-adrenergic receptor activation inhibited N- and P/Q-type calcium currents in medullary raphe neurons via pertussis toxin-sensitive G proteins. In parallel experiments, however, we found that norepinephrine had no effect on G protein-coupled inwardly-rectifying K+ channels in any raphe neurons tested, despite the robust activation of those channels in the same neurons by 5-hydroxytryptamine. Together, these data indicate that alpha 2-adrenergic receptors can modulate N- and P/Q-type calcium channels in caudal medullary raphe neurons but do not couple to the G protein-coupled inwardly-rectifying K+ channels which are also present in those cells. This is in contrast to the effect of 5-hydroxytryptamine1A receptor activation in caudal raphe neurons, and indicates a degree of specificity in the signalling by different pertussis toxin-sensitive G protein-coupled receptors to voltage-dependent calcium channels and G protein-coupled inwardly-rectifying K+ channels even within the same cell system.     

Modulation of high-voltage activated Ca2+ channels in the rat periaqueductal gray neurons by mu-type opioid agonist

The effect of mu-type opioid receptor agonist, D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO), on high-voltage-activated (HVA) Ca2+ channels in the dissociated rat periaqueductal gray (PAG) neurons was investigated by the use of nystatin-perforated patch recording mode under voltage-clamp condition. Among 188 PAG neurons tested, the HVA Ca2+ channels of 38 neurons (32%) were inhibited by DAMGO (DAMGO-sensitive cells), and the other 80 neurons (68%) were not affected by DAMGO (DAMGO-insensitive cells). The N-, P-, L-, Q-, and R-type Ca2+ channel components in DAMGO-insensitive cells shared 26.9, 37.1, 22.3, 7.9, and 5.8%, respectively, of the total Ca2+ channel current. The channel components of DAMGO-sensitive cells were 45.6, 25.7, 21.7, 4.6, and 2.4%, respectively. The HVA Ca2+ current of DAMGO-sensitive neurons was inhibited by DAMGO in a concentration-, time-, and voltage-dependent manner. Application of omega-conotoxin-GVIA occluded the inhibitory effect of DAMGO approximately 70%. So, HVA Ca2+ channels inhibited by DAMGO were mainly the N-type Ca2+ channels. The inhibitory effect of DAMGO on HVA Ca2+ channels was prevented almost completely by the pretreatment of pertussis toxin (PTX) for 8-10 h, suggesting that DAMGO modulation on N-type Ca2+ channels in rat PAG neurons is mediated by PTX-sensitive G proteins. These results indicate that mu-type opioid receptor modulates N-type HVA Ca2+ channels via PTX-sensitive G proteins in PAG neurons of rats.     

Inhibition of M-type K+ and N-type Ca2+ channels by the human gonadotropin-releasing-hormone receptor heterologously expressed in adult neurons

Gonadotropin-releasing hormone (GnRH) controls all aspects of reproductive function. GnRH is secreted by hypothalamic neurons and exerts its effects on the endocrine system through pituitary gonadotropes, while its effects on sexual receptivity are mediated by the central nervous system. The electrophysiological responses of central neurons to GnRH have shown both excitatory and inhibitory responses, but little is known about the mechanisms by which GnRH can change neuronal excitability. The present study addresses the mechanisms whereby stimulation of the human GnRH receptor changes neuronal excitability by using a combination of electrophysiological and heterologous expression techniques. Microinjection of in vitro transcribed cRNA coding for the human GnRH receptor into enzymatically dissociated adult rat superior cervical ganglion neurons resulted in GnRH receptor expression. Activation of the GnRH receptor inhibited both M-type K+ and N-type Ca2+ channels. Inhibition of M-type K+ channels was insensitive to pertussis toxin pretreatment and blocked by intracellular GDPbetaS. Inhibition of Ca2+ channels was slow in onset, voltage independent and insensitive to pertussis toxin. Wash-out of GnRH resulted in an unusual transient reversal of tonic G-protein-mediated Ca2+ channel inhibition. Block of the N-type Ca2+ channel with omega-conotoxin GVIA decreased Ca2+ current inhibition from 43 to 15%, indicating that the N-type Ca2+ channel is an effector target. Ca2+ channel inhibition was completely abolished by including a Ca2+ chelator in the patch pipette. Cell-attached macropatch experiments indicated that Ca2+ channel inhibition is mediated by a diffusible second messenger. These results demonstrate that the human GnRH receptor can inhibit M-type K+ and N-type Ca2+ channels when heterologously expressed in adult rat neurons. Modulation of M-type K+ and N-type Ca2+ channels in central neurons which contain GnRH receptors is likely to contribute to the changes in neuronal excitability elicited by GnRH.     

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. II. Evidence for direct binding

The present study was designed to obtain evidence for direct interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels, using synthetic peptides and fusion proteins derived from loop 1 (cytoplasmic loop between repeat I and II) and the C terminus of these channels. For N-type, prepulse facilitation as mediated by Gbeta gamma was impaired when a synthetic loop 1 peptide was applied intracellularly. Receptor agonist-induced inhibition of N-type as mediated by Galpha was also impaired by the loop 1 peptide but only when applied in combination with a C-terminal peptide. For P/Q-type channels, by contrast, the Galpha-mediated inhibition was diminished by application of a C-terminal peptide alone. Moreover, in vitro binding analysis for N- and P/Q-type channels revealed direct interaction of Galpha with C-terminal fusion proteins as well as direct interaction of Gbeta gamma with loop 1 fusion proteins. These findings define loop 1 of N- and P/Q-type Ca2+ channels as an interaction site for Gbeta gamma and the C termini for Galpha.     

Modulation of [3H]acetylcholine release from cultured amacrine-like neurons by adenosine A1 receptors

We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine-like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+-dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Nucleotide receptors

Adenosine 5'-triphosphate (ATP) and/or related nucleotides act at both ionotropic (P2X) and metabotropic (P2Y) receptors. P2X receptor subunits (P2X1-P2X7) form ligand-gated cation channels, as homomultimers or heteromultimers. Recent work indicates that P2X3 subunits participate in channels expressed by nociceptive sensory neurons, and that the second of the two transmembrane domains of each subunit contributes to the ion permeation pathway. P2X7 subunits form large cytolytic pores in addition to cation channels; they have been found in macrophages and brain microglia. P2Y receptors form a distinct subset of G-protein-coupled receptors; most couple through G proteins to phospholipase C, but inhibition of adenylate cyclase and N-type Ca2+ channels, and activation of K+ channels also occurs. Expressed P2Y receptors have generally been distinguished pharmacologically by the rank order of effectiveness of agonists; some prefer pyrimidines to purines. Recent studies suggest that it is important to use purified nucleotides in such classifications. Several P2Y receptors have a very widespread tissue distribution.     

Photoperiodic history and hypothalamic control of prolactin secretion before birth

Many neuromodulators inhibit N-type Ca2+ currents via G protein-coupled pathways in acutely isolated superior cervical ganglion (SCG) neurons. Less is known about which neuromodulators affect release of norepinephrine (NE) at varicosities and terminals of these neurons. To address this question, we used carbon fiber amperometry to measure catecholamine secretion evoked by electrical stimulation at presumed sites of high terminal density in cultures of SCG neurons. The pharmacological properties of action potential-evoked NE release paralleled those of N-type Ca2+ channels: Release was completely blocked by Cd2+ or omega-conotoxin GVIA, reduced 50% by 10 microM NE or 62% by 2 microM UK-14,304, an alpha2-adrenergic agonist, and reduced 63% by 10 microM oxotremorine M (Oxo-M), a muscarinic agonist. Consistent with action at M2 or M4 receptor subtypes, Oxo-M could be antagonized by 10 microM muscarinic antagonists methoctramine and tropicamide but not by pirenzepine. After overnight incubation with pertussis toxin, inhibition by UK-14,304 and Oxo-M was much reduced. Other neuromodulators known to inhibit Ca2+ channels in these cells, including adenosine, prostaglandin E2, somatostatin, and secretin, also depressed secretion by 34-44%. In cultures treated with omega-conotoxin GVIA, secretion dependent on L-type Ca2+ channels was evoked with long exposure to high K+ Ringer's solution. This secretion was not sensitive to UK-14,304 or Oxo-M. Evidently, many neuromodulators act on the secretory terminals of SCG neurons, and the depression of NE release at terminals closely parallels the membrane-delimited inhibition of N-type Ca2+ currents in the soma.     

Speed of Ca2+ channel modulation by neurotransmitters in rat sympathetic neurons

We have measured the onset and recovery speed of inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion neurons by somatostatin (SS), norepinephrine (NE), and oxotremorine-M (oxo-M, a muscarinic agonist), using the whole cell configuration of the patch-clamp method with 5 mM external Ca2+. With a local perfusion pipette system that changed the solution surrounding the cell within 50 ms, we applied agonists at various times before a brief depolarization from -80 mV that elicited I(Ca). At concentrations that produced maximal inhibition, the onset time constants for membrane-delimited inhibition by SS (0.5 microM), NE (10 microM), and oxo-M (20 microM) were 2.1, 0.7, and 1.0 s, respectively. The time constants for NE inhibition depended only weakly on the concentration, ranging from 1.2 to 0.4 s in the concentration range from 0.5 to 100 microM. Inhibition by oxo-M (20 microM) through a different G-protein pathway that uses a diffusible cytoplasmic messenger had a time constant near 9 s. The recovery rate constant from membrane-delimited inhibition was between 0.09 and 0.18 s(-1), significantly higher than the intrinsic GTPase rate of purified G protein Go, suggesting that Ca2+ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the rate of channel reinhibition after relief by strong depolarizing prepulses, which should reflect the kinetics of final steps in the inhibition process. In the presence of different concentrations of NE, reinhibition was four to seven times faster than the onset of inhibition, indicating that the slowest step of inhibition must precede the binding of G protein to the channel. We propose a kinetic model for the membrane-delimited NE inhibition of Ca2+ channels. It postulates two populations of receptors with different affinities for NE, a single population of G proteins, and a single population of Ca2+ channels. This model closely simulated the time courses of onset and recovery of inhibition and reinhibition, as well as the dose-response curve for inhibition of Ca2+ channels by NE.     

Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals

Ca2+ channels in distinct subcellular compartments of neurons mediate voltage-dependent Ca2+ influx, which integrates synaptic responses, regulates gene expression, and initiates synaptic transmission. Antibodies that specifically recognize the alpha1 subunits of class A, B, C, D, and E Ca2+ channels have been used to investigate the localization of these voltage-gated ion channels on spinal motor neurons, interneurons, and nerve terminals of the adult rat. Class A P/Q-type Ca2+ channels were present mainly in a punctate pattern in nerve terminals located along the cell bodies and dendrites of motor neurons. Both smooth and punctate staining patterns were observed over the surface of the cell bodies and dendrites with antibodies to class B N-type Ca2+ channels, indicating the presence of these channels in the cell surface membrane and in nerve terminals. Class C and D L-type and class E R-type Ca2+ channels were distributed mainly over the cell soma and proximal dendrites. Class A P/Q-type Ca2+ channels were present predominantly in the presynaptic terminals of motor neurons at the neuromuscular junction. Occasional nerve terminals innervating skeletal muscles from the hindlimb were labeled with antibodies against class B N-type Ca2+ channels. Staining of the dorsal laminae of the rat spinal cord revealed a complementary distribution of class A and class B Ca2+ channels in nerve terminals in the deeper versus the superficial laminae. Many of the nerve terminals immunoreactive for class B N-type Ca2+ channels also contained substance P, an important neuropeptide in pain pathways, suggesting that N-type Ca2+ channels are predominant at synapses that carry nociceptive information into the spinal cord.     

Regulators of G protein signaling (RGS) proteins constitutively activate Gbeta gamma-gated potassium channels

Here we report novel effects of regulators of G protein signaling (RGS) on G protein-regulated ion channels. RGS3 and RGS4 induced a substantial increase in currents through the Gbeta gamma-regulated inwardly rectifying K+ channels, IK(ACh), in the absence of receptor activation. Concomitantly, the amount of current that could be activated by agonist was reduced. Pretreatment with pertussis toxin or a muscarinic receptor antagonist abolished agonist-induced currents but did not modify RGS effects. Cotransfection of cells with a Gbetagamma-binding protein significantly reduced the RGS4-induced basal IK(ACh) currents. The RGS proteins also modified the properties of another Gbeta gamma effector, the N-type Ca2+ channels. These observations strongly suggest that RGS proteins increase the availability of Gbeta gamma in addition to their previously described GTPase-activating function.     

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination

Interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels were investigated using the Xenopus oocyte expression system. Gi3alpha was found to inhibit both N- and P/Q-type channels by receptor agonists, whereas Gbeta1 gamma2 was responsible for prepulse facilitation of N-type channels. L-type channels (alpha1C) were not regulated by Galpha or Gbeta gamma. For N-type, prepulse facilitation mediated via Gbeta gamma was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the alpha1C loop 1. Galpha-mediated inhibitions were also impaired by substitution of the alpha1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished Galpha-mediated inhibition. Moreover, a chimera of L-type with the alpha1B loop 1 gained Gbeta gamma-dependent facilitation, whereas an L-type chimera with the N- or P/Q-type C terminus gained Galpha-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for Gbeta gamma and the C termini of P/Q- and N-types for Galpha.     

Dynorphin A-mediated reduction in multiple calcium currents involves a G(o) alpha-subtype G protein in rat primary afferent neurons

We examined the effect of antisera directed at specific G-protein subtype(s) on dynorphin A (Dyn A)-mediated reduction of calcium currents in rat dorsal root ganglia (DRG) neurons. Whole cell patch-clamp recordings were performed on acutely dissociated neurons. Dyn A (1 microM)-mediated decrease in calcium currents was inhibited > 90% by the preferential kappa-receptor antagonist norbinaltorphimine. Dyn A (300-1,000 nM)-mediated reduction in calcium currents was examined during intracellular administration of antisera directed against specific regions of G(o) alpha, G(i) 1 alpha/G(1) 2 alpha, and G(i) 3 alpha subunits. Intracellular dialysis with an antiserum specific for G(o) alpha for 20 min decreased calcium current inhibition by Dyn A (1 microM) in 13 of 15 neurons by an average of 75%. Dialysis with nonimmune serum did not affect Dyn A's action to reduce calcium currents. Intracellular dialysis with either anti-G(i) 1 alpha/G(i) 2 alpha or anti-G(i) 3 alpha antisera did not affect Dyn A-induced changes in calcium currents. In the presence of the N-type calcium channel antagonist omega-conotoxin GVIA, the P-type calcium channel antagonist omega-Aga IVA, and omega-Aga MVIIC applied subsequent to the other toxins, the effect of Dyn A to reduce calcium currents was inhibited by 52, 28, and 16%, respectively. The L channel antagonist nifedipine did not affect the ability to Dyn A to inhibit calcium currents. These results suggest that in rat DRG neurons coupling of kappa-opioid receptors to multiple transient, high-threshold calcium currents involves the G(o) alpha subclass of G proteins.     

Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat

The effect of diabetes mellitus on opiate-mediated inhibition of calcium current density (I(D Ca) [pA pF-1]) and cytosolic calcium response ([Ca2+]i nM) to depolarization with elevated KCl and capsaicin was assessed. Experiments were performed on isolated, acutely dissociated dorsal root ganglion (DRG) neurons from diabetic, BioBreeding/Worcester (BB/W) rats and age-matched control animals. Sciatic nerve conduction velocity was significantly decreased in diabetic animals compared to controls. Mean I(DCa) and [Ca2+]i responses to capsaicin and elevated KCl recorded in DRGs from diabetic animals were significantly larger than those recorded in DRG neurons from controls. In neurons from diabetic animals, the opiate agonist dynorphin A (Dyn A; 1, 3, and 5 microM) had significantly less inhibitory effect on I(D Ca) and KCl-induced [Ca2+]i responses compared to controls. Omega-conotoxin GVIA (omega-CgTX; 10 microM) and pertussis toxin (PTX; 250 ng ml-1) abolished Dyn A-mediated inhibition of I(DCa) and [Ca2+]i in control and diabetic neurons, suggesting that Dyn A modulated predominantly N-type calcium channels coupled to opiate receptors via PTX-sensitive (Gi/o) inhibitory G proteins. These results suggest that opiate-mediated regulation of PTX-sensitive, G protein-coupled calcium channels is diminished in diabetes and that this correlates with impaired regulation of cytosolic calcium.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+ channel/syntaxin interaction

Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the alpha1 subunit. This binding region was loaded into embryonic spinal neurons of Xenopus by early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca2+ concentrations. The relative transmitter release of injected neurons was reduced by approximately 25% at physiological Ca2+ concentration, whereas injection of the corresponding region of the L-type Ca2+ channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. Our data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca2+ dependence to higher values.     

Group I metabotropic glutamate receptors mediate slow inhibition of calcium current in neocortical neurons

Metabotropic glutamate receptor (mGluR)-mediated inhibition of high-voltage-activated Ca2+ currents was investigated in pyramidal neurons acutely isolated from rat dorsal frontoparietal neocortex. Whole cell recordings were made at 30-32 degrees C, with Ca2+ as the charge carrier. Selective agonists were used to classify the subgroup of mGluRs mediating the response. Ca2+ currents were inhibited by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD) and by the group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) but not by the group II agonist (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV) or the group III agonist (+)-2-amino-4-phosphonobutryic acid (-AP4). (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (-CCG-I) was effective at 10 and 100 microM but not at 1 microM, consistent with involvement of group I mGluRs. Variable results were obtained with the putative mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) and the putative mGluR1-selective antagonist (S)-4-carboxyphenylglycine [(S)-4CPG], indicating that the group I mGluR subtypes may vary between cells or that these compounds were activating other receptors. The actions of (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG] were consistent with it being a low-potency antagonist. Several features of the Ca2+ current inhibition evoked by DHPG distinguished it from the rapid modulation typical of a direct action of G proteins on Ca2+ channels; the inhibition was slow to reach maximum (tens of seconds), current activation was not slowed or shifted in the positive voltage direction, and the inhibition was not relieved by positive prepulses. Nimodipine and omega-conotoxin GVIA blocked fractions of the current and also reduced the magnitude of the responses to DHPG, indicating that both L- and N-type Ca2+ channels were regulated. These results further differentiate the slow modulatory pathway observed in neocortical neurons when Ca2+ is used as the charge carrier from the rapid voltage-dependent mechanism reported to inhibit Ba2+ currents under Ca2+-free conditions.