Modulation Of N-type Calcium Channels Research Articles

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Identification and characterization of CIP98 as a novel regulator of mGluR6

Group III metabotropic glutamate receptors (mGluRs) –mGluR4, 6, 7 and 8 – couple exclusively to Gαi/o proteins. mGluR6 has a unique role as the primary postsynaptic neurotransmitter receptor in retinal ON bipolar cells, which receive glutamatergic input directly from photoreceptor cells. Activated mGluR6 initiates a signaling cascade through Gαo that results in inhibition of a cation channel and hyperpolarization of ON bipolar cells. To identify potential regulators of mGluR6 signaling, yeast two‐hybrid screens were developed using intracellular regions of the receptor as baits against a rat retinal library. CIP98 (CASK interacting protein, ‘whirlin’) was identified from these screens. CIP98 has 3 PDZ domains and a proline‐rich domain, but little is known about CIP98 function. We verified expression of CIP98 in ON bipolar cells using single cell RT‐PCR. Using a reconstitution system in sympathetic neurons from the rat superior cervical ganglion (SCG), where we have shown mGluR6 modulation of N‐type calcium channels, we found a dose‐dependent shift in the mGluR6 response to agonist when co‐expressed with a CIP98 truncation mutant. Endogenous CIP98 expression in cultured SCG neurons and hippocampal neurons was confirmed, and knock‐down of endogenous CIP98 with siRNA was used to further examine the impact of CIP98 on mGluR6 function. Together, our data suggest that CIP98 directly regulates mGluR6 signaling.

G Protein-Gated Inhibitory Module of N-Type (CaV2.2) Ca2+ Channels

Voltage-dependent G protein (Gbetagamma) inhibition of N-type (CaV2.2) channels supports presynaptic inhibition and represents a central paradigm of channel modulation. Still controversial are the proposed determinants for such modulation, which reside on the principal alpha1B channel subunit. These include the interdomain I-II loop (I-II), the carboxy tail (CT), and the amino terminus (NT). Here, we probed these determinants and related mechanisms, utilizing compound-state analysis with yeast two-hybrid and mammalian cell FRET assays of binding among channel segments and G proteins. Chimeric channels confirmed the unique importance of NT. Binding assays revealed selective interaction between NT and I-II elements. Coexpressing NT peptide with Gbetagamma induced constitutive channel inhibition, suggesting that the NT domain constitutes a G protein-gated inhibitory module. Such inhibition was limited to NT regions interacting with I-II, and G-protein inhibition was abolished within alpha1B channels lacking these NT regions. Thus, an NT module, acting via interactions with the I-II loop, appears fundamental to such modulation.

A Single Gβ Subunit Locus Controls Cross-talk between Protein Kinase C and G Protein Regulation of N-type Calcium Channels

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gbetagamma subunits in a G protein beta-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gbetagamma target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gbeta(1)-mediated responses. The molecular mechanisms that control this G protein beta subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent approximately 20-amino acid regions of the Gbeta subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gbetagamma signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase beta(2), and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gbeta(1) subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel alpha(1) subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel alpha(1) subunit.

Agonist-independent modulation of N-type calcium channels by ORL1 receptors.

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel-ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.

Molecular determinants of cysteine string protein modulation of N-type calcium channels.

Cysteine string proteins (CSPs) are secretory vesicle chaperones that are important for neurotransmitter release. We have previously reported an interaction of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels that results in a tonic G protein inhibition of the channels. In this report we directly demonstrate that two separate regions of CSP associate with G proteins. The N-terminal binding site of CSP, which includes the J domain, binds Galpha subunits but not Galphabeta subunits whereas the C terminal binding site of CSP associates with either free Galphabeta subunits or Galphabeta in complex with Galpha. The interaction of either binding site of CSP (CSP1-82 or CSP83-198) with G proteins elicits robust tonic inhibition of N-type calcium channel activity. However, CSP1-82 inhibition and CSP83-198 inhibition of calcium channels occur through distinct mechanisms. Calcium channel inhibition by CSP83-198 (but not CSP1-82) is completely blocked by co-expression of the synaptic protein interaction site (synprint) of the N-type channel, indicating that CSP83-198 inhibition is dependent on a physical interaction with the calcium channel. These results suggest that distinct binding sites of CSP can play a role in modulating G protein function and G protein inhibition of calcium channels.

Activation of group II mGlu receptors inhibits voltage-gated Ca2+ currents in myenteric neurons.

The enteric nervous system (ENS) contains functional ionotropic and group I metabotropic glutamate (mGlu) receptors. In this study, we determined whether enteric neurons express group II mGlu receptors and the effects of mGlu receptor activation on voltage-gated Ca(2+) currents in these cells. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a group II mGlu receptor agonist, reversibly suppressed the Ba(2+) current in myenteric neurons isolated from the guinea pig ileum. Significant inhibition was also produced by L-glutamate and the group II mGlu receptor agonists, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I), with a rank order potency of 2R,4R-APDC > DCG-IV > L-glutamate > L-CCG-I, and was reduced by the group II mGlu receptor antagonist LY-341495. Pretreatment of neurons with pertussis toxin (PTX) reduced the action of mGlu receptor agonists, suggesting participation of G(i)/G(o) proteins. Finally, omega-conotoxin GVIA blocked current suppression by DCG-IV, suggesting modulation of N-type calcium channels. mGlu2/3 receptor immunoreactivity was displayed by neurons in culture and in the submucosal and myenteric plexus of the ileum. A subset of these cells displayed a glutamatergic phenotype as shown by the expression of vesicular glutamate transporter 2. These results provide the first evidence for functional group II mGlu receptors in the ENS and show that these receptors are PTX sensitive and negatively coupled to N-type calcium channels. Inhibition of N-type calcium channels produced by activation of group II mGlu receptors may modulate enteric neurotransmission.

Cross-talk between G-protein and Protein Kinase C Modulation of N-type Calcium Channels Is Dependent on the G-protein β Subunit Isoform

The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the alpha(1B) calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195-6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein beta subunits on both wild type and mutant alpha(1B) calcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of Gbeta(1) on channel activity, whereas Gbeta(2), Gbeta(3), and Gbeta(4) are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the Gbeta(1) subunit.

Cysteine String Protein Regulates G Protein Modulation of N-Type Calcium Channels

Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a “J domain” and a palmitoylated cysteine-rich “string” region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca 2+ channels. G α subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, G βγ subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca 2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca 2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.

Calcium Channel β Subunit Promotes Voltage-Dependent Modulation of α1B by G βγ

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming α1 subunit and auxiliary subunits, including the intracellular β subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G βγ dimer concentration. Here we have examined the interaction between the regulation of N-type ( α1B) channels by their β subunits and by G βγ dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC β subunit, we found a significantly larger G βγ-dependent inhibition of α1B channel activation when the VDCC α1B and β subunits were coexpressed. In the absence of coexpressed VDCC β subunit, the G βγ dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC β subunit coexpression restored all of the hallmarks of G βγ modulation. These results suggest that the VDCC β subunit must be present for G βγ to induce voltage-dependent modulation of N-type calcium channels.

G Protein Modulation of N-type Calcium Channels Is Facilitated by Physical Interactions between Syntaxin 1A and Gβγ

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.

Modulation of Neuronal Voltage-gated Calcium Channels by Farnesol

The modulation of presynaptic voltage-dependent calcium channels by classical second messenger molecules such as protein kinase C and G protein betagamma subunits is well established and considered a key factor for the regulation of neurotransmitter release. However, little is known of other endogenous mechanisms that control the activity of these channels. Here, we demonstrate a unique modulation of N-type calcium channels by farnesol, a dephosphorylated intermediate of the mammalian mevalonate pathway. At micromolar concentrations, farnesol acts as a relatively non-discriminatory rapid open channel blocker of all types of high voltage-activated calcium channels, with a mild specificity for L-type channels. However, at 250 nM, farnesol induces an N-type channel-specific hyperpolarizing shift in channel availability that results in approximately 50% inhibition at a typical neuronal resting potential. Additional experiments demonstrated the presence of farnesol in the brain (rodents and humans) at physiologically relevant concentrations (100-800 pmol/g (wet weight)). Altogether, our results indicate that farnesol is a selective, high affinity inhibitor of N-type Ca(2+) channels and raise the possibility that endogenous farnesol and the mevalonate pathway are implicated in neurotransmitter release through regulation of presynaptic voltage-gated Ca(2+) channels.

Identification of an Integration Center for Cross-talk between Protein Kinase C and G Protein Modulation of N-type Calcium Channels

The modulation of presynaptic calcium channel activity by second messengers provides a fine tuning mechanism for neurotransmitter release. In neurons, the activation of certain G protein-coupled receptors reduces N-type channel activity by approximately 60%. In contrast, activation of protein kinase C (PKC) results in an approximately 50% increase in N-type channel activity, and subsequent G protein inhibition is antagonized. Here, we describe the molecular determinants that control the dual effects of PKC-dependent phosphorylation. The double substitution of two adjacent PKC consensus sites in the calcium channel domain I-II linker (Thr422, Ser425) to alanines abolished both PKC-dependent up-regulation and the PKC-G protein cross-talk. The single substitution of Ser425 to glutamic acid abolished PKC up-regulation but had no effect on G protein modulation. Replacement of Thr422 with glutamic acid eliminated PKC-dependent up-regulation and mimicked the effects of PKC phosphorylation on G protein inhibition. Our data suggest that Thr422 mediates the antagonistic effect of PKC on G protein modulation, while phosphorylation of either Thr422 or Ser425 are sufficient to increase N-type channel activity. Thus, Thr422 serves as a molecular switch by which PKC is able to simultaneously trigger the up-regulation of channel activity and antagonize G protein inhibition.

Voltage-dependent modulation of N-type calcium channels: role of G protein subunits.

Voltage-dependent modulation of N-type calcium channels: role of G protein subunits.

G-Protein β-Subunit Specificity in the Fast Membrane-Delimited Inhibition of Ca2+Channels

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.

Regulatory Roles for GTP-Binding Proteins in Nerve Terminals

During the past few years, some roles of GTP-binding proteins in the presynaptic terminal have started to be elucidated. Small GTP-binding proteins, such as rab3a and dynamin, are essential for controlling distinct steps in the cycling of synaptic vesicles, while heterotrimeric G proteins regulate calcium channels, potassium channels, and the secretory machinery either directly or through second messenger pathways. The recent demonstration that G protein-mediated modulation of N-type calcium channels of the presynaptic terminal requires intact syntaxin suggests that G protein regulation of neurotransmitter release may also show an interesting context dependence. Interesting new twists in the G protein story are just emerging.

Voltage-dependent modulation of N-type calcium channels by G-protein beta gamma subunits.

The most commonly used signal transduction pathway for receptor-mediated N-type Ca2+-channel modulation involves activation of a heterotrimeric G protein to produce voltage-dependent inhibition. Although it is widely assumed that Galpha mediates this effect, experiments to address this hypothesis directly are lacking. Here I show that transient overexpression of Gbetagamma in sympathetic neurons mimics and occludes the voltage-dependent Ca2+ channel modulation produced by noradrenaline (NA). Conversely, over-expression of Galpha produces minimal effects on basal Ca2+ channel behaviour but attenuates NA-mediated inhibition in a manner consistent with the buffering of Gbetagamma. These observations indicate that it is Gbetagamma, and Galpha, that mediates voltage-dependent inhibition of N-type Ca2+ channels. The identification of Gbetagamma as the mediator of this pathway has broad implications as G-protein-coupled receptors, many of which are implicated in disease or are targets of therapeutic agents, couple to N-type Ca2+ channels and may modulate synaptic transmission by this mechanism.

Modulation of N-type calcium channels in bullfrog sympathetic neurons by luteinizing hormone-releasing hormone: kinetics and voltage dependence

Inhibition of Ca channel current by luteinizing hormone-releasing hormone (LHRH) was studied in freshly dissociated bullfrog sympathetic ganglion neurons using whole-cell recording. LHRH inhibited up to 80% of the high-threshold Ca channel current with a half-maximally effective concentration of about 20 nM. LHRH inhibited omega-conotoxin-sensitive but not nimodipine-sensitive current and also did not inhibit Bay K 8644-enhanced currents, suggesting that LHRH inhibits N-type but not L-type channels. Inhibition was faster at higher concentrations of LHRH, reaching a limiting time constant of 2 sec at 0.3-3 mM LHRH. The rate of recovery from block (tau approximately 19 sec) was independent of LHRH concentration. Inhibition of N-type current by LHRH was highly sensitive to the gating state of the channel. Though strongly effective if applied when channels were mostly in the resting state, LHRH had little effect if applied rapidly during a long depolarization that opened the channels. Inhibition could be relieved if channels were activated by short, large test depolarizations or by long, smaller depolarizations. The state-dependent properties of LHRH block could be simulated by a model that assumes that inhibition by LHRH results from activated G-proteins binding to N-type channels and that (1) G-protein binding stabilizes closed gating states and (2) activation of G-protein-bound channels destabilizes the binding of the G-protein to the channel.