FMRP regulates presynaptic localization of neuronal voltage gated calcium channels.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism, results from the loss of fragile X mental retardation protein (FMRP). We have recently identified a direct interaction of FMRP with voltage-gated Ca2+ channels that modulates neurotransmitter release. In the present study we used a combination of optophysiological tools to investigate the impact of FMRP on the targeting of voltage-gated Ca2+ channels to the active zones in neuronal presynaptic terminals. We monitored Ca2+ transients at synaptic boutons of dorsal root ganglion (DRG) neurons using the genetically-encoded Ca2+ indicator GCaMP6f tagged to synaptophysin. We show that knock-down of FMRP induces an increase of the amplitude of the Ca2+ transient in functionally-releasing presynaptic terminals, and that this effect is due to an increase of N-type Ca2+ channel contribution to the total Ca2+ transient. Dynamic regulation of CaV2.2 channel trafficking is key to the function of these channels in neurons. Using a CaV2.2 construct with an α-bungarotoxin binding site tag, we further investigate the impact of FMRP on the trafficking of CaV2.2 channels. We show that forward trafficking of CaV2.2 channels from the endoplasmic reticulum to the plasma membrane is reduced when co-expressed with FMRP. Altogether our data reveal a critical role of FMRP on localization of CaV channels to the presynaptic terminals and how its defect in a context of FXS can profoundly affect synaptic transmission.

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits.

CaV1 and CaV2 voltage-gated calcium channels are associated with ß and α2δ accessory subunits. However, examination of cell surface-associated CaV2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged CaV2 channels. Here we report the development of fully functional CaV2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although α2δ subunits copurify with CaV2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α2δ subunits are associated with these channels at the cell surface. We found that, whereas α2δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with CaV2.2/ß1b, despite our finding that α2δ-1 increases plasma-membrane CaV2.2 expression. However, this was due to occlusion of the antigenic epitope by association with CaV2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α2δ-1 and the α1 subunit at the plasma membrane. We further show that, although CaV2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α2δ-1 (but not a gabapentin-insensitive α2δ-1 mutant), the interaction between CaV2.2 and α2δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that CaV2.2 and α2δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction.

Fragile X mental retardation protein controls ion channel expression and activity.

Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (Kv 3.1 and Kv 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary ß4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Cav 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders.

α2δ-1 gene deletion affects somatosensory neuron function and delays mechanical hypersensitivity in response to peripheral nerve damage.

The α2δ-1 subunit of voltage-gated calcium channels is upregulated after sensory nerve injury and is also the therapeutic target of gabapentinoid drugs. It is therefore likely to play a key role in the development of neuropathic pain. In this study, we have examined mice in which α2δ-1 gene expression is disrupted, to determine whether α2δ-1 is involved in various modalities of nociception, and for the development of behavioral hypersensitivity after partial sciatic nerve ligation (PSNL). We find that naive α2δ-1(-/-) mice show a marked behavioral deficit in mechanical and cold sensitivity, but no change in thermal nociception threshold. The lower mechanical sensitivity is mirrored by a reduced in vivo electrophysiological response of dorsal horn wide dynamic range neurons. The CaV2.2 level is reduced in brain and spinal cord synaptosomes from α2δ-1(-/-) mice, and α2δ-1(-/-) DRG neurons exhibit lower calcium channel current density. Furthermore, a significantly smaller number of DRG neurons respond to the TRPM8 agonist menthol. After PSNL, α2δ-1(-/-) mice show delayed mechanical hypersensitivity, which only develops at 11 d after surgery, whereas in wild-type littermates it is maximal at the earliest time point measured (3 d). There is no compensatory upregulation of α2δ-2 or α2δ-3 after PSNL in α2δ-1(-/-) mice, and other transcripts, including neuropeptide Y and activating transcription factor-3, are upregulated normally. Furthermore, the ability of pregabalin to alleviate mechanical hypersensitivity is lost in PSNL α2δ-1(-/-) mice. Thus, α2δ-1 is essential for rapid development of mechanical hypersensitivity in a nerve injury model of neuropathic pain.

Functional remodeling of presynaptic voltage-gated calcium channels in superficial layers of the dorsal horn during neuropathic pain.

N- and P/Q-type voltage-gated Ca2+ channels are critical for synaptic transmission. While their expression is increased in the dorsal root ganglion (DRG) neuron cell bodies during neuropathic pain conditions, less is known about their synaptic remodeling. Here, we combined genetic tools with 2-photon Ca2+ imaging to explore the functional remodeling that occurs in central presynaptic terminals of DRG neurons during neuropathic pain. We imaged GCaMP6s fluorescence responses in an ex vivo spinal cord preparation from mice expressing GCaMP6s in Trpv1-Cre lineage nociceptors. We show that Ca2+ transient amplitude is increased in central terminals of these neurons after spared nerve injury, and that this increase is mediated by both N- and P/Q-type channels. We found that GABA-B receptor-dependent inhibition of Ca2+ transients was potentiated in the superficial layer of the dorsal horn. Our results provide direct evidence toward nerve injury-induced functional remodeling of presynaptic Ca2+ channels in Trpv1-lineage nociceptor terminals.

Stargazin-related protein γ7 is associated with signalling endosomes in superior cervical ganglion neurons and modulates neurite outgrowth.

The role(s) of the newly discovered stargazin-like γ-subunit proteins remains unclear; although they are now widely accepted to be transmembrane AMPA receptor regulatory proteins (TARPs), rather than Ca²âº channel subunits, it is possible that they have more general roles in trafficking within neurons. We previously found that γ7 subunit is associated with vesicles when it is expressed in neurons and other cells. Here, we show that γ7 is present mainly in retrogradely transported organelles in sympathetic neurons, where it colocalises with TrkA-YFP, and with the early endosome marker EEA1, suggesting that γ7 localises to signalling endosomes. It was not found to colocalise with markers of the endoplasmic reticulum, mitochondria, lysosomes or late endosomes. Furthermore, knockdown of endogenous γ7 by short hairpin RNA transfection into sympathetic neurons reduced neurite outgrowth. The same was true in the PC12 neuronal cell line, where neurite outgrowth was restored by overexpression of human γ7. These findings open the possibility that γ7 has an essential trafficking role in relation to neurite outgrowth as a component of endosomes involved in neurite extension and growth cone remodelling.

T-type Ca²âº signalling downregulates MEK1/2 phosphorylation and cross-talk with the RAAS transcriptional response in cardiac myocytes.

Cardiac dysfunction is often associated with an increase in the activity of the renin-angiotensin II-aldosterone system (RAAS). Here, we highlight the cross-talk between the Ca(2+) signalling generated by cardiac T-type current (I(CaT)) and RAAS signalling. Neonatal rat cardiomyocytes exposed to aldosterone, angiotensin II or aldosterone plus angiotensin II co-treatment (AA) show an increase in I(CaT) density, with no cumulative effect of the AA co-treatment. AA increases the amount of T-type channel Ca(v)3.1 mRNA in a time-dependent manner. Angiotensin II increases Ca(v)3.1 mRNA stability, whereas aldosterone increases the transcriptional activity of the Ca(v)3.1 gene promoter. However, in AA-treated cells, angiotensin II decreases aldosterone-induced promoter activity, and aldosterone decreases angiotensin II-induced mRNA stability. The mitogen-activated protein kinase kinase (MEK1/2), which is synergically phosphorylated in AA-treated cells, alters the translocation of glucocorticoid receptors (GR) into the nucleus and attenuates aldosterone-induced promoter activity. In contrast, MEK1/2 has no effect on the NFkB-induced increase in Ca(v)3.1 mRNA and MEK1/2 promoted CREB-target gene transcription. Aldosterone and AA-induced I(CaT) signalling result in a time-dependent activation of the phosphatase PP2A, which dephosphorylates MEK1/2 and CREB. Finally, angiotensin II alone also activates PP2A, which targets MEK1/2, but this activation is independent of I(CaT) calcium signalling and has no effect on CREB phosphorylation. In conclusion, our data demonstrate the cross-talk between a GR-mediated aldosterone response, angiotensin II and the I(CaT) signalling pathways and identify MEK1/2 as a point of connection. This cross-talk results in the fine control of GR- and/or CREB-target gene expression.

Beta-subunits promote the expression of Ca(V)2.2 channels by reducing their proteasomal degradation.

The ß-subunits of voltage-gated calcium channels regulate their functional expression and properties. Two mechanisms have been proposed for this, an effect on gating and an enhancement of expression. With respect to the effect on expression, ß-subunits have been suggested to enhance trafficking by masking an unidentified endoplasmic reticulum (ER) retention signal. Here we have investigated whether, and how, ß-subunits affect the level of Ca(V)2.2 channels within somata and neurites of cultured sympathetic neurons. We have used YFP-Ca(V)2.2 containing a mutation (W391A), that prevents binding of ß-subunits to its I-II linker and found that expression of this channel was much reduced compared with WT CFP-Ca(V)2.2 when both were expressed in the same neuron. This effect was particularly evident in neurites and growth cones. The difference between the levels of YFP-Ca(V)2.2(W391A) and CFP-Ca(V)2.2(WT) was lost in the absence of co-expressed ß-subunits. Furthermore, the relative reduction of expression of Ca(V)2.2(W391A) compared with the WT channel was reversed by exposure to two proteasome inhibitors, MG132 and lactacystin, particularly in the somata. In further experiments in tsA-201 cells, we found that proteasome inhibition did not augment the cell surface Ca(V)2.2(W391A) level but resulted in the observation of increased ubiquitination, particularly of mutant channels. In contrast, we found no evidence for selective retention of Ca(V)2.2(W391A) in the ER, in either the soma or growth cones. In conclusion, there is a marked effect of ß-subunits on Ca(V)2.2 expression, particularly in neurites, but our results point to protection from proteasomal degradation rather than masking of an ER retention signal.

Synthesis of phosphoantigens: scalable accesses to enantiomers of BrHPP and studies on N-HDMAPP synthesis.

Phosphoantigens enable the access to a new anti-tumoral and anti-infectious therapeutic pathway, based on innate immunity through the selective activation of Tγ9δ2 lymphocytes. The first proof of concept of this new immunotherapy approach was demonstrated with the synthetic phosphoantigen named bromohydrin pyrophosphate (BrHPP, IPH 1101) which was administrated in racemic form to about 200 patients in six clinical trials with good safety and promising early signals of efficacy in type C viral hepatitis and follicular non-Hodgkin's lymphoma. Enantiopure samples of BrHPP in gram scale are required for further studies on structure-bioactivity relationship. Thus we developed two complementary synthetic pathways, the first using transformation of a chiral compound and the second involving asymmetric synthesis starting from a prochiral building-block. The synthesis of a second-generation phosphoantigen, N-HDMAPP, which bears a phosphoramidate moiety, was also investigated.

RAR-alpha: Creator, protector, and tormentor.

Retinoic acid receptors are important for homeostatic synaptic plasticity and have many beneficial effects within the brain. New work by Cao et al.1 uncovers a role for these receptors in driving neuropathic pain development, thus identifying a potential preventative therapeutic target.

CaVß-subunit dependence of forward and reverse trafficking of CaV1.2 calcium channels.

Auxiliary CaVß subunits interact with the pore forming CaVα1 subunit to promote the plasma membrane expression of high voltage-activated calcium channels and to modulate the biophysical properties of Ca2+ currents. However, the effect of CaVß subunits on channel trafficking to and from the plasma membrane is still controversial. Here, we have investigated the impact of CaVß1b and CaVß2a subunits on plasma membrane trafficking of CaV1.2 using a live-labeling strategy. We show that the CaVß1b subunit is more potent in increasing CaV1.2 expression at the plasma membrane than the CaVß2a subunit and that this effect is not related to modification of intracellular trafficking of the channel (i.e. neither forward trafficking, nor recycling, nor endocytosis). We conclude that the differential effect of CaVß subunit subtypes on CaV1.2 surface expression is likely due to their differential ability to protect CaV1.2 from degradation.

The life cycle of voltage-gated Ca2+ channels in neurons: an update on the trafficking of neuronal calcium channels.

Neuronal voltage-gated Ca2+ (CaV) channels play a critical role in cellular excitability, synaptic transmission, excitation-transcription coupling and activation of intracellular signaling pathways. CaV channels are multiprotein complexes and their functional expression in the plasma membrane involves finely tuned mechanisms, including forward trafficking from the endoplasmic reticulum (ER) to the plasma membrane, endocytosis and recycling. Whether genetic or acquired, alterations and defects in the trafficking of neuronal CaV channels can have severe physiological consequences. In this review, we address the current evidence concerning the regulatory mechanisms which underlie precise control of neuronal CaV channel trafficking and we discuss their potential as therapeutic targets.

A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels.

A novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

The de novo CACNA1A pathogenic variant Y1384C associated with hemiplegic migraine, early onset cerebellar atrophy and developmental delay leads to a loss of Cav2.1 channel function.

CACNA1A pathogenic variants have been linked to several neurological disorders including familial hemiplegic migraine and cerebellar conditions. More recently, de novo variants have been associated with severe early onset developmental encephalopathies. CACNA1A is highly expressed in the central nervous system and encodes the pore-forming CaVα1 subunit of P/Q-type (Cav2.1) calcium channels. We have previously identified a patient with a de novo missense mutation in CACNA1A (p.Y1384C), characterized by hemiplegic migraine, cerebellar atrophy and developmental delay. The mutation is located at the transmembrane S5 segment of the third domain. Functional analysis in two predominant splice variants of the neuronal Cav2.1 channel showed a significant loss of function in current density and changes in gating properties. Moreover, Y1384 variants exhibit differential splice variant-specific effects on recovery from inactivation. Finally, structural analysis revealed structural damage caused by the tyrosine substitution and changes in electrostatic potentials.

The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin.

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit alpha(2)delta-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both alpha(2)delta ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of alpha(2)delta-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, alpha(2)delta-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of alpha(2)delta-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of alpha(2)delta-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of alpha(2)delta-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of alpha(2)delta-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

The stargazin-related protein gamma 7 interacts with the mRNA-binding protein heterogeneous nuclear ribonucleoprotein A2 and regulates the stability of specific mRNAs, including CaV2.2.

The role(s) of the novel stargazin-like gamma-subunit proteins remain controversial. We have shown previously that the neuron-specific gamma7 suppresses the expression of certain calcium channels, particularly Ca(V)2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of gamma7 on Ca(V)2.2 expression is via an increase in the degradation rate of Ca(V)2.2 mRNA and hence a reduction of Ca(V)2.2 protein level. Furthermore, exogenous expression of gamma7 in PC12 cells also decreased the endogenous Ca(V)2.2 mRNA level. Conversely, knockdown of endogenous gamma7 with short-hairpin RNAs produced a reciprocal enhancement of Ca(V)2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed gamma7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of gamma7 is essential for all its effects, and we show that gamma7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in Ca(V)2.2 mRNA, and is associated with native Ca(V)2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances Ca(V)2.2 I(Ba), and this enhancement is prevented by a concentration of gamma7 that alone has no effect on I(Ba). The effect of gamma7 is selective for certain mRNAs because it had no effect on alpha2delta-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in Ca(V)2.2 mRNA. Our results indicate that gamma7 plays a role in stabilizing Ca(V)2.2 mRNA.

Regulation of T-type Cav3.1 channels expression by synthetic glucocorticoid dexamethasone in neonatal cardiac myocytes.

The effect of the dexamethasone (Dex) on the regulation of the T-type Ca(2+) channel expressions was investigated in primary cultures of neonatal rat ventricular myocytes. We found that Dex (1 microM) increases the T-type Ca(2+) current (I(CaT)) associated with an increase in Ca(v)3.1 mRNA amount. We isolated the upstream region from Ca(v)3.1 encoding gene and tested the activity of the promoter in transfected ventricular myocytes. We found a minimal Dex-responsive region that displayed putative glucocorticoid receptor (GR) and nuclear factor kappa-B (NFkappaB) targets. The GR selective antagonist, RU38486 (10 microM), nearly turned off the transcriptional activity of Ca(v)3.1 encoding gene, and an NFkappaB inhibitor, pyrrolodine dithiocarbonate (10 microM), completely abolished the Dex-induced mRNA increase. However, Dex-induced GR and NFkappaB synthesis and nuclear translocation were not timely related to Ca(v)3.1 mRNA increase. These results indicate that both GR and NFkappaB were necessary, but not sufficient, to trigger the increase in Ca(v)3.1 mRNA amount. This study showed the relationship between glucocorticoid and T-type channels up-regulation that may be involved in cardiac development and pathology.

Disruption of the Key Ca2+ Binding Site in the Selectivity Filter of Neuronal Voltage-Gated Calcium Channels Inhibits Channel Trafficking.

Voltage-gated calcium channels are exquisitely Ca2+ selective, conferred primarily by four conserved pore-loop glutamate residues contributing to the selectivity filter. There has been little previous work directly measuring whether the trafficking of calcium channels requires their ability to bind Ca2+ in the selectivity filter or to conduct Ca2+. Here, we examine trafficking of neuronal CaV2.1 and 2.2 channels with mutations in their selectivity filter and find reduced trafficking to the cell surface in cell lines. Furthermore, in hippocampal neurons, there is reduced trafficking to the somatic plasma membrane, into neurites, and to presynaptic terminals. However, the CaV2.2 selectivity filter mutants are still influenced by auxiliary α2δ subunits and, albeit to a reduced extent, by ß subunits, indicating the channels are not grossly misfolded. Our results indicate that Ca2+ binding in the pore of CaV2 channels may promote their correct trafficking, in combination with auxiliary subunits. Furthermore, physiological studies utilizing selectivity filter mutant CaV channels should be interpreted with caution.

Proteolytic maturation of α2δ controls the probability of synaptic vesicular release.

Auxiliary α2δ subunits are important proteins for trafficking of voltage-gated calcium channels (CaV) at the active zones of synapses. We have previously shown that the post-translational proteolytic cleavage of α2δ is essential for their modulatory effects on the trafficking of N-type (CaV2.2) calcium channels (Kadurin et al., 2016). We extend these results here by showing that the probability of presynaptic vesicular release is reduced when an uncleaved α2δ is expressed in rat neurons and that this inhibitory effect is reversed when cleavage of α2δ is restored. We also show that asynchronous release is influenced by the maturation of α2δ-1, highlighting the role of CaV channels in this component of vesicular release. We present additional evidence that CaV2.2 co-immunoprecipitates preferentially with cleaved wild-type α2δ. Our data indicate that the proteolytic maturation increases the association of α2δ-1 with CaV channel complex and is essential for its function on synaptic release.