Signaling Complexes of Voltage-Gated Calcium Channels and G Protein-Coupled Receptors

Activation of opioid or opioid-receptor-like (ORL1 a.k.a. NOP or orphanin FQ) receptors mediates analgesia through inhibition of N-type calcium channels in dorsal root ganglion (DRG) neurons (). Unlike the three types of classical μ, δ, and κ opioid receptors, ORL1 mediates an agonist-independent inhibition of N-type calcium channels. This is mediated via the formation of a physical protein complex between the receptor and the channel, which in turn allows the channel to effectively sense a low level of constitutive receptor activity (). Further inhibition of N-type channel activity by activation of other G protein-coupled receptors is thus precluded. ORL1 receptors, however, also undergo agonist-induced internalization into lysosomes, and channels thereby become cointernalized in a complex with ORL1. This then results in removal of N-type channels from the plasma membrane and reduced calcium entry (). Similar signaling complexes between N-type channels and GABA_B receptors have been reported (). Moreover, both L-type and P/Q-type channels appear to be able to associate with certain types of G protein-coupled receptors (). Hence, interactions between receptors and voltage-gated calcium channels may be a widely applicable means to optimize receptor channel coupling.     

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of μ-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.     

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

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

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

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

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.     

A single Gbeta 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.     

G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma

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.     

Adenosine inhibits calcium channel currents via A1 receptors on salamander retinal ganglion cells in a mini-slice preparation

The effects of adenosine on high-voltage-activated calcium channel currents in tiger salamander retinal ganglion cells were investigated in a mini-slice preparation. Adenosine produced a concentration-dependent decrease in the amplitude of calcium channel current with a maximum inhibition of 26%. The effects of adenosine on calcium channel current were both time- and voltage-dependent. In cells dialyzed with GTP-gamma-s, adenosine caused a sustained and irreversible inhibition of calcium channel current, suggesting involvement of a GTP-binding protein. The inhibitory effect of adenosine on calcium channel current was blocked by the A1 antagonist 8-cyclopentyltheophylline (DPCPX, 1-10 microm), but not by the A2 antagonist 3-7-dimethyl-1-propargylxanthine (DMPX, 10 microm), and was mimicked by the A1 agonist N6-cyclohexyladenosine (CHA, 1 microm) but not by the A2 agonist 5'-(N-cyclopropyl) carbox-amidoadenosine (CPCA, 1 microm). Adenosine's inhibition of calcium channel current was not affected by the L-type calcium channel blocker nifedipine (5 microm). However, adenosine's inhibition of calcium channel current was reduced to approximately 10% after application of omega-conotoxin GVIA (1 microm), suggesting that adenosine inhibits N-type calcium channels. These results show that adenosine acts on an A1 adenosine receptor subtype via a G protein-coupled pathway to inhibit the component of calcium channel current carried in N-type calcium channels.     

Cross-talk between G-protein and protein kinase C modulation of N-type calcium channels is dependent on the G-protein beta 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.     

G protein-induced trafficking of voltage-dependent calcium channels

Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.     

The cysteine string protein multimeric complex

Cysteine string protein (CSPalpha) is a member of the cellular folding machinery that is located on regulated secretory vesicles. We have previously shown that CSPalpha in association with Hsc70 (70kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) is a guanine nucleotide exchange factor (GEF) for G(alphas). Association of this CSPalpha complex with N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis, triggers tonic G protein inhibition of the channels. Syntaxin 1A, a plasma membrane SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) critical for neurotransmission, coimmunoprecipitates with the CSPalpha/G protein/N-type calcium channel complex, however the significance of syntaxin 1A as a component of this complex remains unknown. In this report, we establish that syntaxin 1A interacts with CSPalpha, Hsc70 as well as the synaptic protein interaction (synprint) region of N-type channels. We demonstrate that huntingtin(exon1), a putative biologically active fragment of huntingtin, displaces both syntaxin 1A and CSPalpha from N-type channels. Identification of the protein components of the CSPalpha/GEF system is essential in establishing its precise role in synaptic transmission.     

Opioid elevation of intracellular free calcium: possible mechanisms and physiological relevance

Opioid receptors are seven transmembrane domain Gi/G0 protein-coupled receptors, the activation of which stimulates a variety of intracellular signalling mechanisms including activation of inwardly rectifying potassium channels, and inhibition of both voltage-operated N-type Ca2+ channels and adenylyl cyclase activity. It is now apparent that like many other Gi/G0-coupled receptors, opioid receptor activation can significantly elevate intracellular free Ca2+ ([Ca2+]i), although the mechanism underlying this phenomenon is not well understood. In some cases opioid receptor activation alone appears to elevate [Ca2+]i, but in many cases it requires concomitant activation of Gq-coupled receptors, which themselves stimulate Ca2+ release from intracellular stores via the inositol phosphate pathway. Given the number of Ca2+-sensitive processes known to occur in cells, there are therefore a myriad of situations in which opioid receptor-mediated elevations of [Ca2+](i) may be important. Here, we review the literature documenting opioid receptor-mediated elevations of [Ca2+]i, discussing both the possible mechanisms underlying this phenomenon and its potential physiological relevance.     

Novel inhibition of gbetagamma-activated potassium currents induced by M(2) muscarinic receptors via a pertussis toxin-insensitive pathway

G(i) protein-coupled receptors such as the M(2) muscarinic acetylcholine receptor (mAChR) and A(1) adenosine receptor have been shown to activate G protein-activated inwardly rectifying K(+) channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M(2) receptors not only activate but also reversibly inhibit these K(+) currents when stimulated with agonist for up to 2 min. The M(2) mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5'-O-(thiotriphosphate) in the pipette. Under these conditions the M(2) mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gbetagamma, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gbetagamma subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M(2) mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M(2) mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M(4) mAChR and alpha(1A) adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A(1) and A(3) adenosine receptors and alpha(2A) and alpha(2C) adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M(2) and M(4) mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gbetagamma-activated GIRK currents in a membrane-delimited fashion.     

Calcium channel beta subunits differentially regulate the inhibition of N-type channels by individual Gbeta isoforms

The direct inhibition of N- and P/Q-type calcium channels by G protein betagamma subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein beta subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. (2000) J. Physiol. (Lond.) 527, 203-212; Cooper, C. B., Arnot, M. I., Feng, Z.-P., Jarvis, S. E., Hamid, J., Zamponi, G. W. (2000) J. Biol. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel beta subunits to modulate the inhibition of alpha(1B) N-type calcium channels by the five known different Gbeta subunit subtypes. Our data reveal that the degree of inhibition by a particular Gbeta subunit is strongly dependent on the specific calcium channel beta subunit, with N-type channels containing the beta(4) subunit being less susceptible to Gbetagamma-induced inhibition. The calcium channel beta(2a) subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For Gbeta(3)-mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the beta(2a) N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel beta subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel beta subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.     

Coordination of membrane excitability through a GIRK1 signaling complex in the atria

Control of heart rate is a complex process that integrates the function of multiple G protein-coupled receptors and ion channels. Among them, the G protein-regulated inwardly rectifying K+ (GIRK or KACh) channels of sinoatrial node and atria play a major role in beat-to-beat regulation of the heart rate. The atrial KACh channels are heterotetrameric proteins that consist of two pore-forming subunits, GIRK1 and GIRK4. Following m2-muscarinic acetylcholine receptor (M2R) stimulation, KACh channel activation is conferred by the direct binding of G protein betagamma subunits (Gbetagamma) to the channel. Here we show that atrial KACh channels are assembled in a signaling complex with Gbetagamma, G protein-coupled receptor kinase, cyclic adenosine monophosphate-dependent protein kinase, two protein phosphatases, PP1 and PP2A, receptor for activated C kinase 1, and actin. This complex would enable the KACh channels to rapidly integrate beta-adrenergic and M2R signaling in the membrane, and it provides insight into general principles governing spatial integration of different transduction pathways. Furthermore, the same complex might recruit protein kinase C (PKC) to the KACh channel following alpha-adrenergic receptor stimulation. Our electro-physiological recordings from single atrial KACh channels revealed a potent inhibition of Gbetagamma-induced channel activity by PKC, thus validating the physiological significance of the observed complex as interconnecting site where signaling molecules congregate to execute a coordinated control of membrane excitability.     

Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region

We have introduced a series of point mutations into the human opioid receptor-like (ORL1) receptor and characterized them for their ability to constitutively activate G protein-coupled receptor signalling pathways. Among the 12 mutants generated, mutation at Asn133 (N133W) gave increased basal signalling through three separate pathways. N133W increased the basal activity of G14- and G16-dependent pathways by two- to three-fold. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. In HEK-293 cells stably expressing N133W, signalling through Gi/o-dependent pathways was also observed. Radioligand binding studies revealed that the affinity for nociceptin of the wild-type ORL1 receptor and the N133W mutant do not differ significantly, suggesting that the ligand binding and signalling functions of constitutively active mutants of G protein-coupled receptors are not necessarily intrinsically linked. In conclusion, our results demonstrate that a mutation in the third transmembrane domain is able to increase the basal signalling activity of the human ORL1 receptor.     

Alternative splicing matters: N-type calcium channels in nociceptors

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

Mu-opioid-receptor-mediated inhibition of the N-type calcium-channel current.

The predominant consequences of mu-opioid-receptor activation are depression of both neuronal activity and transmitter release. Mu-Opioid agonists have previously been observed to increase a potassium conductance and to inhibit adenylate cyclase. We now report that activation of mu-opioid receptors directly decreases the N-type calcium-channel current in a differentiated, human neuroblastoma cell line (SH-SY5Y). The coupling between the mu-opioid receptor and the calcium channel involves a pertussis toxin-sensitive G protein and is independent of changes in adenylate cyclase activity. The inhibition of the calcium-channel current is voltage dependent because it is largely overcome by strong membrane depolarization. It is not associated with changes in the kinetics of current inactivation. Therefore, the mu-receptor belongs to the superfamily of G-protein-coupled, inhibitory neurotransmitter receptors which modulate the activity of calcium and potassium channels and adenylate cyclase.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta 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 beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma 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 beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.