Alternative splicing in the C-terminus of CaV2.2 controls expression and gating of N-type calcium channels

N-type Ca_V2.2 calcium channels localize to presynaptic nerve terminals of nociceptors where they control neurotransmitter release. Nociceptive neurons express a unique set of ion channels and receptors important for optimizing their role in transmission of noxious stimuli. Included among these is a structurally and functionally distinct N-type calcium channel splice isoform, Ca_V2.2e[37a], expressed in a subset of nociceptors and with limited expression in other parts of the nervous system. Ca_V2.2[e37a] arises from the mutually exclusive replacement of e37a for e37b in the C-terminus of Ca_V2.2 mRNA. N-type current densities in nociceptors that express a combination of Ca_V2.2e[37a] and Ca_V2.2e[37b] mRNAs are significantly larger compared to cells that express only Ca_V2.2e[37b]. Here we show that e37a supports increased expression of functional N-type channels and an increase in channel open time as compared to Ca_V2.2 channels that contain e37b. To understand how e37a affects N-type currents we compared macroscopic and single-channel ionic currents as well as gating currents in tsA201 cells expressing Ca_V2.2e[37a] and Ca_V2.2e[37b]. When activated, Ca_V2.2e[37a] channels remain open for longer and are expressed at higher density than Ca_V2.2e[37b] channels. These unique features of the Ca_V2.2e[37a] isoform combine to augment substantially the amount of calcium that enters cells in response to action potentials. Our studies of the e37a/e37b splice site reveal a multifunctional domain in the C-terminus of Ca_V2.2 that regulates the overall activity of N-type calcium channels in nociceptors.     

Slow inhibition of N-type calcium channels with GTP gamma S reflects the basal G protein-GDP turnover rate

The inhibition of N-type Ca channels via a G protein pathway is a rapid mechanism for modulating Ca influx. It has been noted, however, that when G proteins are activated by guanosine 5'- O-(3-thiotriphosphate) (GTPgammaS), the speed of inhibition is greatly reduced, despite the pathway having fewer molecular steps. We explored this anomaly in chick dorsal root ganglion neurons by comparing Ca current inhibition using GTPgammaS with application of the G protein receptor agonist noradrenaline. Noradrenaline caused rapid Ca channel inhibition (tau~5 s), contrasting greatly with the ~70-fold slower rate observed with GTPgammaS. Additionally, the slow rate with GTPgammaS could be accelerated to near agonist-induced rates by application of noradrenaline, demonstrating that the inhibition with GTPgammaS was not perfusion limited and that the rate-limiting step was upstream from GTPgammaS binding. Our results suggest that in the absence of noradrenaline, G protein activation by GTPgammaS is impeded by the slow resting turnover of GDP/GTP. The rate at which inhibition develops with GTPgammaS (tau~350 s) is thus a direct and sensitive measure of resting GDP turnover.     

ORL1 receptor-mediated internalization of N-type calcium channels

The inhibition of N-type calcium channels by opioid receptor like receptor 1 (ORL1) is a key mechanism for controlling the transmission of nociceptive signals. We recently reported that signaling complexes consisting of ORL1 receptors and N-type channels mediate a tonic inhibition of calcium entry. Here we show that prolonged ( approximately 30 min) exposure of ORL1 receptors to their agonist nociceptin triggers an internalization of these signaling complexes into vesicular compartments. This effect is dependent on protein kinase C activation, occurs selectively for N-type channels and cannot be observed with mu-opioid or angiotensin receptors. In expression systems and in rat dorsal root ganglion neurons, the nociceptin-mediated internalization of the channels is accompanied by a significant downregulation of calcium entry, which parallels the selective removal of N-type calcium channels from the plasma membrane. This may provide a new means for long-term regulation of calcium entry in the pain pathway.     

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.     

Tonic inhibition of neuronal calcium channels by G proteins removed during whole-cell patch-clamp experiments

The barium current through voltage-dependent calcium channels was recorded from cultured rat cortical neurons with the whole-cell configuration of the patch-clamp technique. The maximal current evoked by depolarising pulses from –80 mV to 0 mV was divided into inactivating and non-inactivating fractions. During the first minutes of whole-cell recording, the amplitude of the inactivating fraction increased from less than 0.1 nA to an average value of 1 nA, whereas the amplitude of the non-inactivating component remained essentially the same. This increase in amplitude was prevented when the perforated-patch technique was used, suggesting that some intracellular factor that inhibited the barium current was lost or destroyed during conventional whole-cell experiments. When GTP[-S] or GTP was added to the pipette solution, no increase or only a weak rise of the inactivating current was seen, whereas GDP[-S] accelerated its increase. The results suggest that some of the calcium channels expressed in cultured cortical neurons are inhibited by a G protein even in the absence of added neurotransmitter. The current increase observed during whole-cell recordings may be due to a loss of intracellular GTP and the subsequent inactivation of an inhibitory G protein.     

N-type voltage-dependent calcium channels mediate the nicotinic enhancement of GABA release in chick brain

The role of voltage-dependent calcium channels (VDCCs) in the nicotinic acetylcholine receptor (nAChR)-mediated enhancement of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) was investigated in chick brain slices. Whole cell recordings of neurons in the lateral spiriform (SpL) and ventral lateral geniculate (LGNv) nuclei showed that cadmium chloride (CdCl2) blocked the carbachol-induced increase of spontaneous GABAergic IPSCs, indicating that VDCCs might be involved. To conclusively show a role for VDCCs, the presynaptic effect of carbachol on SpL and LGNv neurons was examined in the presence of selective blockers of VDCC subtypes. omega-Conotoxin GVIA, a selective antagonist of N-type channels, significantly reduced the nAChR-mediated enhancement of gamma-aminobutyric acid (GABA) release in the SpL by 78% compared with control responses. Nifedipine, an L-type channel blocker, and omega-Agatoxin-TK, a P/Q-type channel blocker, did not inhibit the enhancement of GABAergic IPSCs. In the LGNv, omega-Conotoxin GVIA also significantly reduced the nAChR-mediated enhancement of GABA release by 71% from control values. Although omega-Agatoxin-TK did not block the nicotinic enhancement, L-type channel blockers showed complex effects on the nAChR-mediated enhancement. These results indicate that the nAChR-mediated enhancement of spontaneous GABAergic IPSCs requires activation of N-type channels in both the SpL and LGNv.     

Scanning mutagenesis reveals a role for serine 189 of the heterotrimeric G-protein beta 1 subunit in the inhibition of N-type calcium channels

Direct interactions between the presynaptic N-type calcium channel and the beta subunit of the heterotrimeric G-protein complex cause voltage-dependent inhibition of N-type channel activity, crucially influencing neurotransmitter release and contributing to analgesia caused by opioid drugs. Previous work using chimeras of the G-protein beta subtypes Gbeta1 and Gbeta5 identified two 20-amino acid stretches of structurally contiguous residues on the Gbeta1 subunit as critical for inhibition of the N-type channel. To identify key modulation determinants within these two structural regions, we performed scanning mutagenesis in which individual residues of the Gbeta1 subunit were replaced by corresponding Gbeta5 residues. Our results show that Gbeta1 residue Ser189 is critical for N-type calcium channel modulation, whereas none of the other Gbeta1 mutations caused statistically significant effects on the ability of Gbeta1 to inhibit N-type channels. Structural modeling shows residue 189 is surface exposed, consistent with the idea that it may form a direct contact with the N-type calcium channel alpha1 subunit during binding interactions.     

Comparative analysis of inactivated-state block of N-type (Cav2.2) calcium channels

Objective

The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays.

Methods

The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K⁺ channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology.

Results

N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC₅₀ values of 0.001?C7???M and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable.

Conclusions

The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.     

Go transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons

High-voltage-activated (HVA) calcium channel currents (I _Ba) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. I _Ba was irreversibly inhibited by 56.9±2.7% by 1 M -conotoxin-GVIA (-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective -aminobutyric acid_B (GABA_B) agonist, (–)-baclofen (50 M), inhibited the HVA I _Ba by 30.7±5.4%. Prior application of -CTx-GVIA completely occluded inhibition of the HVA I _Ba by (–)-baclofen, indicating that in this preparation (–)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G_o antiserum, raised against the C-terminal decapeptide of the G _o subunit, reduced (–)-baclofen inhibition of the HVA I _Ba, whereas replating DRGs in the presence of the anti-G_i antiseram did not. Using anti-G _o antisera (12000) and confocal laser microscopy, G _o localisation was investigated in both unreplated and replated neurons. G _o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G _o immunoreactivity was completely blocked by preincubation with the immunising G_o undecapeptide (1 g · ml^–1) for 1 h at 37° C. A similar treatment also blocked recognition of G _o in brain membranes on immunoblots. These results provide evidence that GABA_B inhibition of N-type calcium channels in acutely replated DRGs occurs via G _o.     

Voltage-gated calcium channels in chronic pain: emerging role of alternative splicing

N- and T-type voltage-gated calcium channels are key established players in chronic pain. Current work suggests that alternative splicing of these channels constitutes an important aspect in the investigation of their roles in the pathogenesis of chronic pain. Recent N-type channel studies describe a nociceptor-enriched alternatively spliced module responsible for voltage-independent G protein modulation and internalization, which is implicated in the control of distinct nociceptive pathways. On the contrary, although a large body of work has demonstrated that peripheral Ca_v3.2-encoded T-type currents are involved in several types of chronic pain, little is known with respect to the expression of numerous newly discovered splice variants in specific pain pathways. The elucidation of the new layers of molecular complexity uncovered in N- and T-type channel splice variants and their respective locations and roles in different pain pathways will allow for the development of better therapeutic strategies for the treatment of chronic pain.     

omega-conotoxin GVIA alters gating charge movement of N-type (CaV2.2) calcium channels

omega-conotoxin GVIA (omegaCTX) is a specific blocker of N-type calcium (CaV2.2) channels that inhibits neuropathic pain. While the toxin appears to be an open channel blocker, we show that N-channel gating charge movement is modulated. Gating currents were recorded from N-channels expressed along with beta2a and alpha2delta subunits in HEK293 cells in external solutions containing either lanthanum and magnesium (La-Mg) or 5 mM Ca2+ plus omegaCTX (omegaCTX-Ca). A comparison showed that omegaCTX induced a 10-mV right shift in the gating charge versus voltage (Q-V) relationship, smaller off-gating current time constant (tau Q(Off)), a lower tau Q(Off) voltage dependence, and smaller on-gating current (Q(On)) tau. We also examined gating current in La-Mg plus omegaCTX and found no significant difference from that in omegaCTX-Ca; this demonstrates that the modulation was induced by the toxin. A model with strongly reduced open-state occupancy reproduced the omegaCTX effect on gating current and showed that the gating modulation alone would inhibit N-current by 50%. This mechanism of N-channel inhibition could be exploited to develop novel analgesics that induce only a partial block of N-current, which may limit some of the side effects associated with the toxin analgesic currently approved for human use (i.e., Prialt).     

Kindling induces a long-term enhancement in the density of N-type calcium channels in the rat hippocampus

How seizures arise and recur in epilepsy is unknown. Recent genetic, pharmacological and electrophysiological data indicate a significant but undisclosed role for voltage-dependent calcium channels. Since the contribution such channels make to nerve function reflects the targeting of discrete subtypes to distinct cellular regions, we hypothesized that epilepsy reflects alterations in their spatiotemporal patterns of expression at the cell surface. To test this possibility, we examined the expression and distribution of hippocampal N-type calcium channels in an animal seizure model: kindling. Confocal microscopy of N-type calcium channels labeled with a new fluorescent ligand, coupled with a novel technique for analysing multiple images, revealed a 20-40% increase in their expression in CA1 and CA3 within 24 h post-seizure. These increases persisted in the dendritic fields of CA1, but had dissipated in CA3 by 28 days post-seizure. Such changes correlate poorly with cell number or synaptogenesis, but are consistent with increased N-type calcium channel expression on presynaptic terminals or, more likely, dendrites. These data rationalize recent electrophysiology and in situ hybridization data, and suggest that kindling alters N-type calcium channel trafficking mechanisms to cause a persistent, local, remodeling of their distributions in CA1 dendrites. The persistent induction of N-type calcium channels may be part of a mechanism for, and a hallmark of, synaptic plasticity, in which kindling represents a reinforcement of synapses en masse.     

Alternative splicing in the voltage-sensing region of N-Type CaV2.2 channels modulates channel kinetics

The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaValpha1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, we generated four tsA201 cell lines stably expressing CaV2.2 splice isoforms. Coexpression of auxiliary CaVbeta and CaValpha2delta subunits was required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activated and deactivated more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaValpha2delta to resolve gating currents. The steady-state voltage dependence of charge movement was not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decayed with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlated reasonably well with the properties of native N-type currents in sympathetic neurons. Our results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating.     

Functional impact of alternative splicing of human T-type Cav3.3 calcium channels

Low-voltage-activated T-type (Cav3) Ca2+ channels produce low-threshold spikes that trigger burst firing in many neurons. The CACNA1I gene encodes the Cav3.3 isoform, which activates and inactivates much more slowly than the other Cav3 channels. These distinctive kinetic features, along with its brain-region-specific expression, suggest that Cav3.3 channels endow neurons with the ability to generate long-lasting bursts of firing. The human CACNA1I gene contains two regions of alternative splicing: variable inclusion of exon 9 and an alternative acceptor site within exon 33, which leads to deletion of 13 amino acids (Delta33). The goal of this study is to determine the functional consequences of these variations in the full-length channel. The cDNA encoding these regions were cloned using RT-PCR from human brain, and currents were recorded by whole cell patch clamp. Introduction of the Delta33 deletion slowed the rate of channel opening. Addition of exon 9 had little effect on kinetics, whereas its addition to Delta33 channels unexpectedly slowed both activation and inactivation kinetics. Modeling of neuronal firing showed that exon 9 or Delta33 alone reduced burst firing, whereas the combination enhanced firing. The major conclusions of this study are that the intracellular regions after repeats I and IV play a role in channel gating, that their effects are interdependent, suggesting a direct interaction, and that splice variation of Cav3.3 channels provides a mechanism for fine-tuning the latency and duration of low-threshold spikes.