Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

doi:10.1038/ncomms4628

Download PDF

Article
Open access
Published: 07 April 2014

Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

Laurent Ferron¹,
Manuela Nieto-Rostro¹^na1,
John S. Cassidy¹^na1 &
…
Annette C. Dolphin¹

Nature Communications volume 5, Article number: 3628 (2014) Cite this article

4888 Accesses
96 Citations
5 Altmetric
Metrics details

Subjects

Abstract

Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (Ca_V) channels. Here we show that the functional expression of neuronal N-type Ca_V channels (Ca_V2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases Ca_V channel density in somata and in presynaptic terminals. We then show that FMRP controls Ca_V2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and Ca_V2.2 occurs between the carboxy-terminal domain of FMRP and domains of Ca_V2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via Ca_V2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

Altered dendritic spine function and integration in a mouse model of fragile X syndrome

Article Open access 23 October 2019

Sam A. Booker, Aleksander P. F. Domanski, … Peter C. Kind

Aberrant activity of mitochondrial NCLX is linked to impaired synaptic transmission and is associated with mental retardation

Article Open access 02 June 2021

Alexandra Stavsky, Ohad Stoler, … Israel Sekler

FMRP(1–297)-tat restores ion channel and synaptic function in a model of Fragile X syndrome

Article Open access 02 June 2020

Xiaoqin Zhan, Hadhimulya Asmara, … Ray W. Turner

Introduction

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability, often associated with autism^1,2. FXS is caused, in the vast majority of cases, by a trinucleotide repeat expansion in the 5′-UTR of the fragile X mental retardation 1 (FMR1) gene, leading to its hypermethylation and transcriptional silencing. The FMR1 gene encodes fragile X mental retardation protein (FMRP), an RNA-binding protein that participates in the trafficking of messenger RNA (mRNAs) to distal sites in neurons, especially dendrites of central neurons³. FMRP suppresses translation of mRNAs to which it binds, but also regulates the activity-dependent translation of these mRNAs, particularly in response to group I metabotropic glutamate receptor activation^4,5. These findings show that dendritic FMRP is critical for postsynaptic activity of neurons⁶.

A growing body of evidence points to an additional role for FMRP in presynaptic function^7,8,9. For example, a recent study showed that Fmr1 knockout mice have abnormal presynaptic short-term plasticity in hippocampal neurons¹⁰. This opened the possibility that there is an effect of FMRP on presynaptic voltage-gated calcium (Ca_V) channels.

The Ca_V family plays a major role in the physiology of excitable cells¹¹. Three subfamilies have been identified: Ca_V1–3. The Ca_V1 (L-type) and Ca_V2 (N-, P/Q- and R-type) channels are thought to be heteromultimers composed of the pore-forming α₁-subunit, associated with auxiliary Ca_Vβ- and Ca_Vα₂δ-subunits¹¹. N-type calcium channels (Ca_V2.2) are present in both the central and peripheral nervous systems, and they have a major presynaptic role in regulation of transmitter release^12,13. The intracellular loop between domains II and III and the C-terminal tail of Ca_V2 α₁-subunits interact with presynaptic proteins that modulate calcium currents and the targeting of these channels to synaptic terminals¹⁴. Since there are precedents for interaction of FMRP with ion channels, specifically the sodium-activated K⁺ channel (Slack-B)¹⁵ and the calcium-activated potassium channel (BK)¹⁶, we examined whether FMRP could directly affect calcium channel function.

Here we show that Ca_V channel density in dorsal root ganglion (DRG) neurons is increased at the cell surface of the soma and the presynaptic terminals when FMRP is knocked down with small hairpin RNA (shRNA). We then show that FMRP reduces Ca_V2.2-generated currents by decreasing the expression of the channel at the plasma membrane, and we provide evidence that this mechanism involves proteasomal degradation. We also show that the interaction between FMRP and Ca_V2.2 is direct, since we find that the C-terminal domain of FMRP binds to the Ca_V2.2 II–III linker and C-terminal domains. Finally, using a pharmacological approach, we show that FMRP modulates synaptic vesicle release via Ca_V2.2 channels. Our data demonstrate an unexpected effect of FMRP on Ca_V2.2 channel functional expression, and indicate a new role for FMRP in presynaptic function.

Results

FMRP regulates Ca_V2.2 expression in DRG neurons

To determine the impact of FMRP on Ca_V currents in DRG neurons, we silenced FMRP expression using RNA interference. Knockdown of FMRP was validated in tsA-201 cells expressing green fluorescent protein (GFP)-FMRP and in cultured DRG neurons (Supplementary Fig. 1). We then examined the effect of FMRP knockdown on calcium currents in DRG neurons 4 days after shRNA transfection (Fig. 1a,b). We found that, in DRG neurons in which FMRP was knocked down, the peak Ca_V current density was increased by 103% (Fig. 1b). Previous work found that N-type calcium currents account for the major component of calcium current in DRG neurons¹⁷. We then examined the impact of FMRP knockdown on Ca_V2.2 surface expression. To do this, we transfected DRG neurons with a Ca_V2.2 channel construct tagged with an exofacial HA epitope and either control shRNA or FMRP shRNA (Fig. 1c). We found that HA-Ca_V2.2 immunoreactivity at the cell surface was increased by 45% in neurons in which FMRP was knocked down (Fig. 1c,d).

**Figure 1: FMRP knockdown increases Ca_V currents and enhances surface expression of Ca_V2.2 channels in soma and presynaptic terminals of DRG neurons.**

We then took advantage of the fact that DRG neurons form functional synapses, when co-cultured with dorsal horn (DH) neurons¹⁸, to investigate the impact of FMRP on Ca_V2.2 expression at presynaptic terminals. P2 DRG neurons were co-microinjected with HA-Ca_V2.2 and the presynaptic protein VAMP-1, tagged with mCherry, together with either control shRNA or FMRP shRNA (Fig. 1e,f). We found that HA-Ca_V2.2 immunoreactivity in VAMP-mCherry-positive boutons in non-permeabilized processes was increased by 48% in neurons in which FMRP was knocked down (Fig. 1g). Finally, we assessed Ca_V2.2 expression in brain synaptosomes from Fmr1 knockout mice (Fig. 1h). We found that the level of Ca_V2.2 protein in synaptosomes was increased by 43% in Fmr1 knockout mice compared with wild-type mice. Together, our data reveal that FMRP controls the presynaptic expression of Ca_V2.2 in DRG neurons.

FMRP controls Ca_V2.2 current density

To characterize the functional effect of FMRP on N-type calcium channels, we recorded Ba²⁺ currents (I_Ba) from tsA-201 cells co-transfected with Ca_V2.2 (together with auxiliary subunits Ca_Vβ1b and Ca_Vα₂δ-1) and GFP-FMRP. We found that FMRP significantly reduced peak Ca_V2.2 I_Ba current density at +10 mV by 80% (Fig. 2a,b).

**Figure 2: FMRP reduces Ca_V2.2 current density.**

A reduction of current density can result from a loss of functional channels at the plasma membrane and/or a modification of the biophysical properties of the channels. No difference in the voltage dependence of activation for the current density–voltage relationships was observed when FMRP was co-expressed with Ca_V2.2 (Fig. 2b) and this was confirmed by tail current analysis (Fig. 2c,d). However, we noted a consistent depolarizing shift of the Ca_V2.2 steady-state inactivation curve when FMRP was co-expressed (Fig. 2e,f). This latter modification is not responsible for the reduction of current density but nevertheless it suggests that FMRP may be interacting with intracellular domains of Ca_V2.2 involved in voltage-dependent inactivation. We also examined the kinetics of decay of the current, but observed no modification of the time constant of inactivation (Fig. 2g). Importantly, the reduction in current density was still evident using the physiological charge carrier, Ca²⁺; FMRP reduced peak Ca_V2.2 I_Ca density at 0 mV by 55% (Fig. 2h).

Since FMRP has been shown to modulate the gating of Slack-B channels¹⁵, we therefore assessed the impact of FMRP on Ca_V2.2 channel activity. Using fluctuation analysis, we found that FMRP does not modify the single-channel conductance of Ca_V2.2 (Fig. 3a–c). Alternatively, a reduction of open probability of the channels could also account for a lower whole-cell current density. We therefore estimated Ca_V2.2 maximum open probability by comparing the maximal ON-gating charge displaced (Q_max) with the whole-cell conductance (G_max) for each cell¹⁹ (Fig. 3d–h). We found that Q_max for Ca_V2.2 was reduced by 38% when FMRP was co-expressed, suggesting there are fewer channels in the plasma membrane; but neither the gating charge kinetics (Fig. 3f) or voltage dependence (Fig. 3g), nor the estimated channel maximum open probability (Fig. 3h) were modified. Taken together, our results indicate that FMRP is likely to act by reducing the number of available Ca_V2.2 channels on the cell surface.

**Figure 3: Effect of FMRP on biophysical properties of Ca_V2.2.**

FMRP reduces cell surface Ca_V2.2 via proteasomal degradation

To test for FMRP regulation of Ca_V2.2 protein expression, we compared Ca_V2.2 protein level in tsA-201 cells transfected with or without FMRP (Fig. 4a). We found that FMRP does not affect total Ca_V2.2 protein level, or expression of the auxiliary subunits (Fig. 4b and Supplementary Fig. 2).

**Figure 4: FMRP reduces Ca_V2.2 cell surface expression.**

Therefore, we assessed plasma membrane expression of Ca_V2.2 using a cell surface-biotinylation assay, and observed that biotinylated Ca_V2.2 was significantly reduced in the presence of FMRP, by 28.7% (Fig. 4c–g). Ca_V2.2 plasma membrane expression was further investigated by immunocytochemistry using an extracellular HA-tagged Ca_V2.2 channel construct (Fig. 4e). We found that HA-Ca_V2.2 immunoreactivity at the cell surface of non-permeabilized cells was reduced by 43.9% when Ca_V2.2 was co-expressed with FMRP (Fig. 4g). The reduction of Ca_V2.2 cell surface expression determined by both methods is in agreement with the reduction of current density that we observed (Fig. 2).

Ca_V channels are subject to degradation by the proteasome^20,21. To test whether FMRP influences the targeting of cell surface-expressed Ca_V2.2 to the proteasome, we examined the effect of the proteasome inhibitor MG132 on Ca_V2.2 protein levels in the absence or presence of FMRP (Fig. 4d–f). We first confirmed that MG132 treatment was effective in blocking the degradation of Ca_V2.2 expressed in tsA-201 cells (total Ca_V2.2 level is increased by 56% in cells treated with MG132 compared with control, Supplementary Fig. 3). Then, using both the biotinylation assay and immunocytochemistry, we found that MG132 treatment abolished the reduction of Ca_V2.2 surface expression induced by FMRP (Fig. 4h). Together, our results indicate that FMRP modulates Ca_V2.2 expression at the plasma membrane by targeting the channels for proteasomal degradation.

FMRP interacts with Ca_V2.2 channels

To determine whether FMRP interacts with Ca_V2.2, we first used immunoprecipitation on whole-cell lysate from tsA-201 cells expressing Ca_V2.2 and HA-FMRP. We found that an HA Ab co-immunoprecipitates Ca_V2.2 together with HA-FMRP (Fig. 5a). The interaction is likely to be directly with Ca_V2.2 because HA-FMRP does not co-immunoprecipitate the co-expressed auxiliary Ca_Vβ1b or Ca_Vα₂δ-1 subunits (Fig. 5a), also pointing to the possibility that it might interact preferentially with channels that are not in functional complexes with their auxiliary subunits. The interaction between Ca_V2.2 and FMRP was confirmed with an in situ proximity ligation assay (PLA). Using GFP Ab to detect GFP-Ca_V2.2 and FMRP Ab to detect HA-FMRP, we found PLA-positive signals only in cells expressing GFP-Ca_V2.2 (Fig. 5b); notably, the signal was present within the cytoplasm, as well as at the plasma membrane.

To identify which domain of FMRP is involved in interaction with Ca_V2.2, we performed pull-down experiments using glutathione S-transferase (GST) fusion proteins with FMRP N-terminal or C-terminal domains (Fig. 5c). We applied whole-cell lysate from tsA-201 cells expressing Ca_V2.2/Ca_Vβ1b/Ca_Vα₂δ-1 to each purified GST-fusion protein, and found that only the C-terminal domain of FMRP pulled down Ca_V2.2 (Fig. 5d). We further showed that the C-terminal domain of FMRP is necessary for its functional effect because FMRP with a deletion of its C-terminal domain is unable to reduce Ca_V2.2 current density (Supplementary Fig. 4). The interaction is specific for Ca_V2.2 because neither Ca_Vβ1b nor Ca_Vα₂δ-1 was pulled down (Fig. 5e). We also found that Ca_V2.2 containing a mutation (W391A) that prevents binding of Ca_Vβ subunits to its I-II linker²² retains its ability to interact with the FMRP C-terminus, further suggesting that the interaction between FMRP and Ca_V2.2 is independent of Ca_Vβ (Fig. 5f). Finally, the interaction of FMRP with Ca_V channels appears to be specific for the Ca_V2 family since Ca_V2.1 but not Ca_V1.2 channels binds to the FMRP C-terminus (Supplementary Fig. 5).

FMRP is phosphorylated primarily on the conserved S499 in its C-terminal domain¹. To test whether this key serine is involved in the interaction with Ca_V2.2, we mutated S499 into either an alanine (S499A, dephosphomimetic)²³ or an aspartic acid (S499D, phosphomimetic)⁵. We performed GST pull-down experiments and found that neither mutation prevented the binding of FMRP C-terminus to Ca_V2.2 (Fig. 5g,h).

FMRP interacts with Ca_V2.2 in vivo

We then examined whether the interaction between FMRP and Ca_V2.2 occurs in neurons. We first showed that FMRP interacts with Ca_V2.2 endogenously, by immunoprecipitation of either Ca_V2.2 or FMRP from brain synaptosomes. We found that a Ca_V2.2 Ab co-immunoprecipitates FMRP (Fig. 6a), and reciprocally we showed that an FMRP Ab co-immunoprecipitates Ca_V2.2 (Fig. 6b). We also confirmed that the FMRP C-terminal domain interacts with synaptosomal Ca_V2.2 (Fig. 6c). FMRP is expressed in DRG neurons throughout the soma and processes²⁴ (Supplementary Fig. 6). To determine whether the interaction between FMRP and Ca_V2.2 occurs in neurons, we transiently transfected DRG neurons with GFP-Ca_V2.2 and performed in situ PLA using anti-GFP Ab and anti-FMRP Ab. We found PLA-positive signals in somata and processes of DRG neurons expressing GFP-Ca_V2.2, showing that endogenous FMRP also forms protein complexes with Ca_V2.2 both in DRG neuron cell bodies and in their neurites (Fig. 6d–f).

**Figure 6: FMRP interacts with endogenous Ca_V2.2 in neurons.**

FMRP interacts with Ca_V2.2 synaptic targeting domains

We then focused on identifying the domains of Ca_V2.2 involved in the interaction with FMRP. We generated a series of GFP-tagged constructs corresponding to intracellular regions of Ca_V2.2 (Fig. 7a). We expressed each construct individually in tsA-201 cells and applied the whole-cell lysate to immobilized GST-FMRP C-terminus (Fig. 7b–g). We found that the II–III linker (Fig. 7d) and the C-terminus of Ca_V2.2 (Fig. 7f) were both pulled down by FMRP C-terminus, but the other intracellular regions were not (Fig. 7b,c,e). Interestingly, these two intracellular regions of Ca_V2.2 are both involved in the interaction with presynaptic proteins²⁵. The II–III linker contains a synaptic protein interaction (synprint)²⁵ site and we found that this synprint motif on its own retains the ability to bind to FMRP C-terminus (Fig. 7g).

**Figure 7: Intracellular domains of Ca_V2.2 determining the interaction with FMRP.**

FMRP controls synaptic release via N-type calcium channels

Ca_V2 channels have a major presynaptic role in regulation of transmitter release^12,13, and Ca_V2.2 is particularly important in the peripheral nervous system. In order to determine the physiological impact of the interaction between FMRP and Ca_V2.2 channels, we assessed the effect of loss of FMRP on synaptic vesicle release. We used the vGlut1-pHluorin (vGpH) optical reporter to measure synaptic vesicle recycling in DRG neurons co-cultured with DH neurons²⁶. E18 DRG neurons were co-transfected with mCherry, vGpH and either Ctrl shRNA or FMRP shRNA and then plated with untransfected DH neurons. DRG neurons were imaged after 9–11 days in culture. vGpH colocalized in varicosities with presynaptic marker synapsin and was in apposition to postsynaptic marker PSD-95 (Fig. 8a). We monitored the increase in fluorescence of vGpH in response to 40 action potentials at 10 Hz (Fig. 8b). Signals from each bouton were normalized to the fluorescence value obtained by rapid alkalinization of the entire labelled vesicle pool using NH₄Cl (Fig. 8b). We found that responses to 40 action potentials at 10 Hz were increased by 37% in synapses from DRG neurons in which FMRP was knocked down compared with control (Fig. 8c–e). During stimulation, the change of vGpH signal reflects the difference between exocytosis and ongoing vesicle endocytosis and reacidification²⁷. After stimulation, the signal decays due to endocytosis of vGpH and vesicle reacidification. We then examined synaptic vesicle recycling by stimulating neurons at 40 Hz for 30 s and monitoring the time course of fluorescence decay. We found that the rate of decay was not different in boutons of DRG neurons lacking FMRP compared with controls (Fig. 8f). We can then conclude that the increase of vesicle release during stimulation of synaptic terminals of which FMRP was knocked down is due to an increase in exocytosis. Interestingly, we recorded a similar increase in vesicle release in synapses from the hippocampal neurons expressing FMRP shRNA (37.5% increase compared with Ctrl shRNA, Supplementary Fig. 7), suggesting that FMRP is also involved in vesicular release in central nervous system synapses.

**Figure 8: FMRP knockdown enhances synaptic vesicle exocytosis in presynaptic terminals of DRG neurons via Ca_V2.2 channels.**

Using specific blockers for Ca_V2.2 and Ca_V2.1 channels (ω-conotoxin GVIA and ω-agatoxin IVA, respectively), we showed that in control synapses 55.3% of the exocytosis was inhibited by ω-conotoxin GVIA and that 82.7% was blocked by ω-conotoxin GVIA+ω-agatoxin IVA co-application (Fig. 8c–e). Remarkably, at synapses of DRG neurons in which FMRP was knocked down, ω-conotoxin GVIA reduced exocytosis to a level comparable with the control (inhibition of 58.4%, Fig. 8e) and therefore abolished the increase induced by the lack of FMRP. Finally, ω-conotoxin GVIA together with ω-agatoxin IVA reduced exocytosis by 82% in DRG neurons in which FMRP was knocked down (Fig. 8e). Together, these data demonstrate that FMRP regulates vesicle exocytosis via Ca_V2.2 channels in DRG neurons.

Discussion

FXS, the most common heritable form of mental retardation, is characterized by synaptic dysfunction and is caused by the loss of FMRP^1,2. Regulation of Ca_V expression at presynaptic terminals is a critical factor in the control of synaptic transmission. In this study, we show that FMRP exerts a tonic inhibition of somatic Ca_V current density and Ca_V2.2 surface expression at presynaptic terminals of DRG neurons. We also show that FMRP reduces Ca_V2.2-generated currents by decreasing the expression of the channels at the plasma membrane, and we provide evidence that this mechanism depends on proteasomal degradation. We demonstrate that the interaction between FMRP and Ca_V2.2 is direct, since we find that the C-terminal domain of FMRP binds to the Ca_V2.2 II–III linker and its C-terminal domain. Finally, we show that FMRP controls synaptic vesicle exocytosis via Ca_V2.2 channels. Our study demonstrates a hitherto unknown effect of FMRP on Ca_V2.2 expression and indicates a major presynaptic function for FMRP.

In this study, we show that FMRP affects Ca_V2.2 cell surface expression at the presynaptic element of DRG neurons and modulates synaptic transmission by this means. Consistent with this hypothesis, studies in animal models of FXS show an increase in transmitter release at the Drosophila neuromuscular junction^7,8 and an increase of synaptic vesicle recycling in mouse hippocampal neurons¹⁰. However, the lack of FMRP has also been associated with decrease of synaptic transmission^7,28,29, and FMRP appears to regulate multiple synaptic parameters depending on the developmental stage and the area of the nervous system.

Ca_V2.1 and Ca_V2.2 channels are the two major classes of Ca_V in presynaptic terminals, whereas Ca_V1.2 channels are located mainly in postsynaptic elements¹⁴. We show that FMRP interacts with Ca_V2.1 and Ca_V2.2, but not with Ca_V1.2 channels, suggesting that the interaction is specific for presynaptic calcium channels (Supplementary Fig. 5). We also show that FMRP interacts with the II–III linker synprint site and C-terminal region of Ca_V2.2. Several presynaptic proteins have been found to interact with Ca_V2.2 channels, including SNARE proteins syntaxin 1A and SNAP-25 (refs 25, 30) and active-zone proteins including Rab-interacting molecule (RIM)³¹. The interaction between SNARE proteins and Ca_V2.2 synprint site has been found to modulate the targeting of the Ca_V2.2 channel to the nerve terminals of both peripheral sympathetic ganglion neurons³² and central hippocampal neurons³³ and also to inhibit calcium currents³⁴. Moreover, Ca_V2.2 channels, via a postsynaptic density-95/discs large/zona occludens-1 (PDZ) domain binding motif and a proline-rich region located in the Ca_V2.2 C-terminal domain, can directly interact with Mint, CASK, RIM1 and RIM-BP2 to target the channels to presynaptic active zones^35,36. G protein-coupled receptor have also been shown to associate with Cav2.2 via the II–III linker and the C-terminal domain³⁷ and this can alter the expression of the Cav2.2 channels at the plasma membrane^38,39. These interactions provide an effective association between Ca²⁺ entry and the vesicle release sites that ensures the rapid triggering of neurotransmitter release when an action potential invades the nerve terminal. Precisely how the interaction with FMRP affects Ca_V2.2 synaptic localization is still unclear and further investigation is required to elucidate this process.

Several mechanisms can control Ca_V expression at the plasma membrane; among them interactions with the auxiliary Ca_Vα₂δ-1 subunits markedly affect their functional expression¹¹. We show that FMRP does not affect Ca_Vα₂δ-1 expression and does not interact with Ca_Vα₂δ-1, making it unlikely that FMRP affects Ca_V2.2 surface expression via a Ca_Vα₂δ subunit-dependent mechanism, despite the role of Ca_Vα₂δ proteins in presynaptic calcium channel targeting²⁶. Trafficking and surface expression of Ca_V also depends on the interaction with auxiliary Ca_Vβ subunits¹¹. Recent studies have shown that the interaction of Ca_V1 and Ca_V2 channels with Ca_Vβ subunits protects these channels against proteasomal degradation^20,21. Interestingly, we find here that the effect of FMRP on Ca_V2.2 expression is antagonized by a proteasome inhibitor, and we found that the interaction between FMRP and Cav2.2 does not co-immunoprecipitate Ca_Vβ subunits, suggesting that it occurs with channels that have lost, or subsequently lose, the protective interaction with Ca_Vβ subunits¹¹. Nevertheless, further work is needed to unravel the molecular mechanism by which FMRP targets Ca_V2.2 for degradation. For example, does FMRP also interact with an element of the proteasome and thus target Ca_V2.2 to this pathway, in which case FMRP would play the role of a molecular adaptor?

Our findings reveal that FMRP interacts directly with the Ca_V2.2 channel protein, both in vitro and in cultured DRG neurons. A recent study has reported that FMRP can directly interact and modulate the gating of the sodium-activated potassium channel Slack-B¹⁵. Whereas in this latter report, the FMRP N-terminal domain is shown to be involved in the interaction with Slack-B, we find here that the FMRP C-terminal domain interacts with Ca_V2.2. The FMRP N-terminal domain is a well-described platform for protein–protein interactions⁴⁰ but only two reports have so far described protein–protein interactions with FMRP C-terminal domain: the motor protein kinesin light chain involved in RNA granule and axonal transport³ and the scaffolding protein Ran-binding protein in the microtubule-organizing centre (RanBPM), which is associated with synapse formation in neurons^41,42. The FMRP C-terminal domain is a non-conserved region in the related proteins FXR1P and FXR2P¹. Altogether, these data reinforce the hypothesis that the FMRP C-terminal domain contributes to determining the specificity of FMRP function⁴¹.

More recently, FMRP has been shown to interact with large-conductance calcium-activated potassium (BK) channel via their auxiliary β4 subunit and to regulate neurotransmitter release at hippocampal CA3-CA1 synapses by modulating action potential duration in the soma of CA3 neurons¹⁶. This latter study also described that the intracellular perfusion of FMRP Ab induces an increase of calcium transient in presynaptic boutons of CA3 neurons, although the mechanism behind this increase of calcium transient was not examined. BK channels are modulators of action potential duration in the soma of CA3 neurons, but their involvement in presynaptic vesicle exocytosis is still a matter of debate⁴³. Further investigation is needed to identify the mechanism by which FMRP modulates the calcium transient in the CA3-CA1 hippocampal synapses but our results support the view of an involvement of Ca_V2 channels.

Taken together, our data demonstrate a direct and tonic role for FMRP in regulating presynaptic N-type calcium channel expression and function in DRG neurons. Presynaptic Ca_V2.2 channels are critical for neurotransmission, both in central neurons and in the autonomic and sensory nervous system, where they are involved in nociception and short-term synaptic plasticity^14,44. Therefore, dysregulation of Ca_V2.2 expression could account for aspects of the cognitive impairment and altered pain sensitivity observed both in patients and in a mouse model of FXS^24,45,46. Consistent with this hypothesis, a clinical trial using a γ-aminobutyric acid type B (GABA_B) agonist (R-baclofen), an inhibitory modulator of N-type calcium channels⁴⁷, found some improvement of social function and behaviour in FXS patients⁴⁸. Our study opens the possibility that small molecule inhibitors of Ca_V2.2, currently being developed for a number of chronic pain conditions⁴⁹, could be of benefit in FXS.

Methods

cDNA constructs

The following cDNAs were used: Ca_V2.2 (D14157), Ca_V2.1 (M64373), Ca_V1.2 (M67515), Ca_Vβ1b (X61394; from Dr T. P. Snutch,), Ca_Vα₂δ-1 (Genbank accession number AF_286488), mut-3b GFP(M62653, except S72A and S65G, Dr T. E. Hughes), all subcloned in pMT2 for expression in tsA-201 cells and subcloned in pcDNA3.0 or pRK5 vectors for expression in neurons. peGFP-FMRP (NBCI nucleotide accession code NM_008031.2) was a gift from Dr G. J. Bassell; Vamp-mCherry and vGlut-pHluorin was a gift from Dr T.A. Ryan. The HA-tagged Ca_V2.2 construct was generated with an HA tag in an extracellular linker, similar to that previously described²⁰. Truncated and GFP-tagged Ca_V2.2 constructs (Nter-GFP, I-II-GFP, GFP-II–III, GFP-synprint, GFP-III–IV and GFP-Cter) and HA-FMRP were generated using standard molecular biological techniques and confirmed by DNA sequencing. All constructs were cloned into the pcDNA3.0 vector for expression in mammalian cells. shRNA plasmids were generated as previously described⁵⁰ using the following mRNA target sequence for FMRP: 5′-GTGATGAAGTTGAGGTTTA-3′ (ref. 51).

Cell culture and transfection

tsA-201 cells (96121229, ECACC) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS), penicillin and streptomycin, and 2% GlutaMAX (Invitrogen). tsA-201 cells were transfected using FuGene6 reagent (Roche and Promega), following the manufacturer’s instructions. P10 DRG neurons were prepared from male Sprague Dawley rats and transfected using Amaxa Nucleofector (Lonza) according to the manufacturer’s protocol. E18 and P2 rat DRG/DH neuron co-cultures were prepared from isolated DRGs and spines. Neurons were dissociated by trypsinization (0.25%) followed by trituration. Neurons were plated on glass coverslips coated with poly-L-lysine and laminin and cultured in Neurobasal medium containing B27 supplement, penicillin and streptomycin, NGF (100 ng ml⁻¹), and 2% GlutaMAX (Invitrogen). One-half of the growth medium was replaced every 3 days. DRG neurons were transfected before plating using Amaxa Nucleofactor (Lonza) according to the manufacturer’s instructions or microinjected (Eppendorf microinjection system) after 7 days in culture.

Western blot analysis

At 48 h after transfection, cells were rinsed twice with phosphate-buffered saline (PBS) and then harvested in PBS containing protease inhibitors (Complete tablet from Roche). The cells were lysed in PBS, 1% Igepal and protease inhibitors for 30 min on ice. The detergent lysates were then clarified by centrifugation (14,000 g, 30 min, 4 °C). Proteins were separated by SDS–PAGE on 3–8% Tris-acetate or 4–12% Bis-Tris gels and then transferred to polyvinylidene fluoride membranes. After blocking in Tris-buffered saline buffer (10 mM Tris, pH 7.4, 500 mM NaCl. 0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with primary antibody overnight. The protein–Ab complexes were then labelled with a horseradish peroxidase-conjugated secondary Ab (1:3,000 Sigma-Aldrich) for 1 h at room temperature and detected using the enhanced ECL Plus reagent (GE Healthcare) visualized with a Typhoon 9410 scanner (GE Healthcare). Quantification of immunoblot bands was performed with ImageQuant software (GE Healthcare). The following Abs were used: rabbit anti-Ca_V2.2 (1:500)⁵², rabbit anti-Ca_Vβ1b (1:500) and mouse anti-Ca_Vα₂δ-1 (1:3,000, D219, Sigma).

Synaptosomal fraction preparation

Synaptosomal fractions were prepared by differential centrifugation⁵³. Whole rat brains were homogenized in: 0.32 M sucrose, 3 mM HEPES-Na, pH 7.4 containing protease inhibitors. The homogenate was centrifuged at 1,000 g for 10 min to produce a pellet (P1) and a supernatant (S1). The pellet P1 was resuspended in homogenization buffer and centrifuged at 1,000g for 10 min to produce a pellet (P1′) and a supernatant (S1′). S1 and S1′ were combined and centrifuged at 12,000 g for 15 min to produce a pellet P2 and a supernatant S2. P2 was resuspended in homogenization buffer and centrifuged 15 min at 13,000 g to produce the crude synaptosomal fraction P2′. P2′ was resuspended in 1% Igepal PBS, incubated for 30 min on ice and clarified by centrifugation 30 min at 12,000 g.

Immunoprecipitation

Clarified cell lysates or synaptosomal fractions were cleared with 50 μg of protein A sepharose (GE Healthcare) for 1 h at 4 °C. Supernatants were incubated with 2 μg ml⁻¹ of specific Ab overnight at 4 °C with constant agitation. A further 20 μg of protein A sepharose was added and incubated for 1 h at 4 °C. Beads were washed three times with PBS containing 0.1% Igepal and incubated for 15 min at 55 °C with 100 mM dithiothreitol and 2X Laemmli sample buffer. Eluted proteins were then resolved by SDS–PAGE. The following Abs were used: rat anti-HA (Roche), MAB 7G1-1 anti-FMRP (Developmental Studies Hybridoma Bank, University of Iowa), MAB2160 anti-FMRP (Millipore) and rabbit anti-Ca_V2.2.

Cell surface biotinylation

At 18 h after transfection, cells were rinsed twice with PBS and then incubated with PBS containing 1 mg ml⁻¹ Sulfo-NHS-SS-Biotin (Perbio) for 30 min at room temperature and then rinsed once with PBS and twice with PBS containing 200 mM glycine. Cells were then harvested and lysed in PBS, 1% Igepal and protease inhibitors for 30 min on ice. The detergent lysates were then clarified by centrifugation (14,000 × g, 30 min, 4 °C). Biotinylated proteins were precipitated by adding 100 μl of streptavidin-agarose beads (Perbio) and incubated overnight at 4 °C. The streptavidin-agarose beads were washed three times and incubated for 1 h at 37 °C with 100 mM dithiothreitol and 2x Laemmli sample buffer. Eluted proteins were then resolved by SDS–PAGE. The following Abs were used: rabbit anti-Ca_V2.2 (1:500)⁵² and rabbit polyclonal anti-Akt (9272, 1:1,000, Cell Signalling Technology).

GST pull-down assay

For pull-down assays, GST was subcloned into pYES2.1/V5-His TOPO TA (Invitrogen) by inserting PCR product using pGEX-2T as a template (GE Healthcare). GST-tagged constructs were generated by inserting PCR products of the mouse FMRP N-terminal (nucleotides 266–1003) and C-terminal (nucleotides 1514–2104) into EcoRI site of a pYES2.1/V5-His-GST. Yeasts (Saccharomyces cerevisiae) were transformed with individual expression vectors encoding the GST-fusion proteins and produced by standard methods. The yeast was lysed by vigorous shaking in PBS containing protease inhibitors (Complete tablet, Roche) and glass beads (Sigma) at 4 °C for 20 min. The lysates were then clarified by centrifugation (14,000 g, 5 min, 4 °C). GST-fusion proteins were immobilized on glutathione sepharose 4B beads (GE Healthcare) and incubated at 4 °C with lysate from tsA-201 cells transfected with full-length Ca_V2.2 or GFP-tagged Ca_V2.2 constructs or synaptosomal fraction preparations. Beads were washed four times with ice-cold 1% Triton-PBS containing protease inhibitors (Complete tablet, Roche) and incubated for 15 min at 55 °C with 100 mM dithiothreitol and 2X Laemmli sample buffer. Eluted proteins were then resolved by SDS–PAGE. The following Abs were used: rabbit polyclonal anti-Ca_V2.2 (ref. 52) (1:500), mouse monoclonal anti-GST (sc-138, 1:3,000, Santa Cruz Biotechnology) and mouse monoclonal anti-GFP (632380, 1:3,000, Clontech).

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde/sucrose in PBS for 5 min and then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were blocked with 10% FBS in PBS for at least 30 min at room temperature, and incubated with the primary Ab overnight with 3% FBS in PBS. Primary antibodies used were as follows: rabbit anti-βIII-tubulin (T2200, 1:1,000, Sigma), rabbit anti-synapsin 1, 2 (106002, 1:200, Synaptic systems), mouse anti-PSD-95 (ab2723, 1:200, Abcam). Samples were then washed and incubated with secondary conjugated Ab (1:500; anti-rabbit AF594, A11072; anti-rabbit AF633, A31573; anti-mouse AF594, A11005; anti-mouse AF633, A21050; all from Invitrogen and anti-mouse FITC, F2012, from Sigma) for 1 h at room temperature. After washing, samples were mounted in VectaShield (Vector Laboratories). Cells were examined on LSM 510 Meta or LSM 780 confocal microscopes (Zeiss).

In situ PLA

Cells were fixed with 4% paraformaldehyde/sucrose in PBS for 5 min, incubated in 0.1 M Tris HCl for 5 min at room temperature and then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were blocked with 10% FBS/0.1% Tween 20 in 4 × SSC for 30 min at 37 °C. Cells were incubated overnight with the primary antibody pair (1:500) from different species, directed against GFP (rabbit polyclonal anti-GFP, TP-401; Torrey Pines) and FMRP (mouse monoclonal MAB 7G1-1, Developmental Studies Hybridoma Bank, University of Iowa) and thereafter subjected to in situ PLA using Duolink Detection kit (Olink Bioscience) according to the manufacturer’s instruction. Briefly, after incubation with primary antibodies, the cells were incubated with secondary antibodies conjugated with oligonucleotides (mouse PLA probe MINUS and rabbit PLA probe PLUS, supplied in the PLA kit). Subsequently, connector oligonucleotides and ligase were added; the connector oligonucleotides hybridize to the two PLA probes and join to form a circular DNA strand when the PLA probes are in close proximity. After ligation, a polymerase is added and rolling-circle amplification is initiated using one of the PLA probes as a primer. The amplification product is detected through hybridization of fluorescently labelled oligonucleotides. Thus, individual pairs of proteins generate a spot that can be visualized using fluorescence microscopy. The theoretical maximum distance between the 2 target proteins is 30–40 nm to be able to create a PLA signal.

Live cell imaging

Coverslips were mounted in a rapid-switching, laminar-flow perfusion and stimulation chamber (Warner Instruments) on the stage of an epifluorescence microscope (Axiovert 200 M, Zeiss). Live cell images were acquired with an Andor iXon+ (model DU-897U-CS0-BV) back-illuminated EMCCD camera using OptoMorph software (Cairn Research, UK). White and 470 nm LEDs served as light sources (Cairn Research, UK). Fluorescence excitation and collection was done through a 40 × 1.3 NA Fluar Zeiss objective using 450/50 nm excitation and 510/50 nm emission and 480 nm dichroic filters (for pHluorin) and a 572/35 nm excitation and low-pass 590 nm emission and 580 nm dichroic filters (for mCherry). Action potentials were evoked by passing 1 ms current pulses via platinum electrodes. Cells were perfused (0.5 ml min⁻¹) in a saline solution at 30 °C containing (in mM) 119 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, 25 HEPES (buffered to pH 7.4), 30 glucose, 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 μM D,L-2-amino-5-phosphonovaleric acid (AP5, Sigma). ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (300 nM) were applied for 10 min before stimulation (Alomone Labs). NH₄Cl applications were done with 50 mM NH₄Cl in substitution of 50 mM of NaCl (buffered to pH 7.4). Images were acquired at 2 Hz and analysed in ImageJ (http://www.rsb.info.nih.gov/ij) using a custom-written plugin (http://www.rsb.info.nih.gov/ij/plugins/time-series.html).

Electrophysiology

Whole-cell patch-clamp recordings were performed on tsA-201 cells or DRG neurons at room temperature (21–25 °C). Single cells were voltage clamped using an Axopatch 200B patch-clamp amplifier (Axon instruments). Patch pipettes were filled with a solution containing the following (in mM): 140 Cs-aspartate, 5 EGTA, 2 MgCl₂, 0.1 CaCl₂, 2 K₂ATP and 10 HEPES, titrated to pH 7.2 with CsOH. The external solution contained the following (in mM): 150 tetraethylammonium bromide, 3 KCl, 1 NaHCO₃, 1 MgCl₂, 10 HEPES, 4 glucose and 1 BaCl₂, pH adjusted to 7.4 with Tris base. Recordings from DRG neurons were performed using 10 mM BaCl₂. Current density–voltage relationships were fitted with a modified Boltzmann equation as follows: (1) I=G_max × (V−V_rev)/(1+exp(−(V−V_50,act)/k)), where I is the current density (in pA/pF), G_max is the maximum conductance (in nS/pF), V_rev is the reversal potential, V_50,act is the midpoint voltage for current activation and k is the slope factor. Steady-state inactivation and activation data were fitted with a single Boltzmann equation of the following form: (2) I/I_max=(A₁−A₂)/[1+exp((V−V_50,inact)/k/]+A₂), where I_max is the maximal current, and V_50,inact is the half-maximal voltage for current inactivation. For the steady-state inactivation, A₁ and A₂ represent the proportion of inactivation and non-inactivating current, respectively. Inactivation kinetics of the currents were estimated by fitting the decaying part of the current traces with the following equation: (3) I(t)=C+A × exp(−(t−t₀)/τ_inact), where t₀ is zero time, C the fraction of non-inactivating current, A the relative amplitude of the exponential, and τ_inact is the time constant.

Non-stationary fluctuation analysis

Fluctuation analysis was carried out using the method described by Sigworth⁵⁴. Ensembles of currents were generated by a series of identical voltage pulses delivered every 5 s. Currents were recorded using 10 mM BaCl₂ and filtered at 2 kHz. The variance was calculated between successive sweeps and then averaged over all the pairs. Background variance at the holding potential was subtracted from the variance during the test pulse. Single-channel current was estimated by plotting the variance as a function of the mean current and fitting the data by (4) variance=i × I−I²/N, where i is the unitary current, I is the mean current and N is the number of functional channels⁵⁵.

Gating currents

ON-gating currents were measured during test pulses to positive potentials at which no ionic inward or outward currents were observed. Currents were filtered at 10 kHz. Patch pipettes were filled with a solution containing the following (in mM): 150 N-methyl-D-glucamine (NMDG), 10 EGTA, 1 MgCl₂, 10 HEPES and 4 Mg-ATP (adjusted to pH 7.3 with methanesulfonic acid). The external solution contained the following (in mM): 135 choline chloride, 4 MgCl₂, 10 Hepes and 1 CaCl₂; pH adjusted to 7.2 with CsOH.

G/Q analysis

G/Q analyses were performed as previously described¹⁹. G_max was determined for each cell as the slope of the peak current–voltage relationship (between +20 mV and +60 mV). G_max is related to single-channel open probability at maximal depolarization by the following equation: (5) G_max=P_Omaxng, where P_Omax is the single-channel open probability at maximal depolarization, n is the number of channels and g is the single-channel conductance. Q_max can be determined according to the following equation: (6) Q_max=nq_max, where q_max is the maximum gating charge moved per single channel. Plotting G_max as a function of Q_max defines a linear relationship with a slope (7) G_max/Q_max=P_Omax(g/q_max). Assuming that the single-channel conductance is not modified (Fig. 3c) and that the number of elementary charges moved are the same (voltage dependence of activation of their gating currents are indistinguishable, Fig. 3g) then the slope G_max/Q_max is proportional to P_Omax.

Statistical analysis

Statistical analysis was conducted by one-way analysis of variance followed by Tukey’s multiple comparison tests. Statistical significance was set at P<0.05, and all error bars represent s.e.m.

Additional information

How to cite this article: Ferron, L. et al. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density. Nat. Commun. 5:3628 doi: 10.1038/ncomms4628 (2014).

Accession codes

Accessions

GenBank/EMBL/DDBJ

AF_286488

References

Bassell, G. J. & Warren, S. T. Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function. Neuron 60, 201–214 (2008).
Article CAS Google Scholar
Bhakar, A. L., Dolen, G. & Bear, M. F. The pathophysiology of fragile X (and what it teaches us about synapses). Annu. Rev. Neurosci. 35, 417–443 (2012).
Article CAS Google Scholar
Dictenberg, J. B., Swanger, S. A., Antar, L. N., Singer, R. H. & Bassell, G. J. A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome. Dev. Cell 14, 926–939 (2008).
Article CAS Google Scholar
Zalfa, F. et al. The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses. Cell 112, 317–327 (2003).
Article CAS Google Scholar
Muddashetty, R. S. et al. Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation, and mGluR signaling. Mol. Cell 42, 673–688 (2011).
Article CAS Google Scholar
Bear, M. F., Huber, K. M. & Warren, S. T. The mGluR theory of fragile X mental retardation. Trends Neurosci. 27, 370–377 (2004).
Article CAS Google Scholar
Zhang, Y. Q. et al. Drosophila fragile X-related gene regulates the MAP1B homolog Futsch to control synaptic structure and function. Cell 107, 591–603 (2001).
Article CAS Google Scholar
Gatto, C. L. & Broadie, K. Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure. Development 135, 2637–2648 (2008).
Article CAS Google Scholar
Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011).
Article CAS Google Scholar
Deng, P. Y., Sojka, D. & Klyachko, V. a. Abnormal presynaptic short-term plasticity and information processing in a mouse model of fragile X syndrome. J. Neurosci. 31, 10971–10982 (2011).
Article CAS Google Scholar
Dolphin, A. C. Calcium channel auxiliary alpha2delta and beta subunits: trafficking and one step beyond. Nat. Rev. Neurosci. 13, 542–555 (2012).
Article CAS Google Scholar
Hirning, L. D. et al. Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons. Science 239, 57–61 (1988).
Article ADS CAS Google Scholar
Turner, T. J., Adams, M. E. & Dunlap, K. Multiple Ca2+ channel types coexist to regulate synaptosomal neurotransmitter release. Proc. Natl Acad. Sci. USA 90, 9518–9522 (1993).
Article ADS CAS Google Scholar
Catterall, W. A. & Few, A. P. Calcium channel regulation and presynaptic plasticity. Neuron 59, 882–901 (2008).
Article CAS Google Scholar
Brown, M. R. et al. Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack. Nat. Neurosci. 13, 819–821 (2010).
Article CAS Google Scholar
Deng, P. Y. et al. FMRP regulates neurotransmitter release and synaptic information transmission by modulating action potential duration via BK channels. Neuron 77, 696–711 (2013).
Article CAS Google Scholar
Nowycky, M. C., Fox, A. P. & Tsien, R. W. Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316, 440–443 (1985).
Article ADS CAS Google Scholar
Hendrich, J., Bauer, C. S. & Dolphin, A. C. Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture. Channels (Austin.) 6, 124–132 (2012).
Article CAS Google Scholar
Agler, H. L. et al. G protein-gated inhibitory module of N-type (ca(v)2.2) ca2+ channels. Neuron 46, 891–904 (2005).
Article CAS Google Scholar
Altier, C. et al. The Cavbeta subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels. Nat. Neurosci. 14, 173–180 (2011).
Article CAS Google Scholar
Waithe, D., Ferron, L., Page, K. M., Chaggar, K. & Dolphin, A. C. Beta-subunits promote the expression of Ca(V)2.2 channels by reducing their proteasomal degradation. J. Biol. Chem. 286, 9598–9611 (2011).
Article CAS Google Scholar
Leroy, J. et al. Interaction via a key tryptophan in the I-II linker of N-type calcium channels is required for beta1 but not for palmitoylated beta2, implicating an additional binding site in the regulation of channel voltage-dependent properties. J. Neurosci. 25, 6984–6996 (2005).
Article CAS Google Scholar
Narayanan, U. et al. FMRP phosphorylation reveals an immediate-early signaling pathway triggered by group I mGluR and mediated by PP2A. J. Neurosci. 27, 14349–14357 (2007).
Article CAS Google Scholar
Price, T. J. et al. Decreased nociceptive sensitization in mice lacking the fragile X mental retardation protein: role of mGluR1/5 and mTOR. J. Neurosci. 27, 13958–13967 (2007).
Article CAS Google Scholar
Sheng, Z. H., Rettig, J., Takahashi, M. & Catterall, W. A. Identification of a syntaxin-binding site on N-type calcium channels. Neuron 13, 1303–1313 (1994).
Article CAS Google Scholar
Hoppa, M. B., Lana, B., Margas, W., Dolphin, A. C. & Ryan, T. A. Alpha2delta expression sets presynaptic calcium channel abundance and release probability. Nature 486, 122–125 (2012).
Article ADS CAS Google Scholar
Sankaranarayanan, S. & Ryan, T. A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nat. Cell Biol. 2, 197–204 (2000).
Article CAS Google Scholar
Suvrathan, A., Hoeffer, C. A., Wong, H., Klann, E. & Chattarji, S. Characterization and reversal of synaptic defects in the amygdala in a mouse model of fragile X syndrome. Proc. Natl Acad. Sci. USA 107, 11591–11596 (2010).
Article ADS CAS Google Scholar
Patel, A. B., Hays, S. A., Bureau, I., Huber, K. M. & Gibson, J. R. A target cell-specific role for presynaptic Fmr1 in regulating glutamate release onto neocortical fast-spiking inhibitory neurons. J. Neurosci. 33, 2593–2604 (2013).
Article CAS Google Scholar
Mochida, S., Sheng, Z. H., Baker, C., Kobayashi, H. & Catterall, W. A. Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca2+ channels. Neuron 17, 781–788 (1996).
Article CAS Google Scholar
Coppola, T. et al. Direct interaction of the Rab3 effector RIM with Ca2+ channels, SNAP-25, and synaptotagmin. J. Biol. Chem. 276, 32756–32762 (2001).
Article CAS Google Scholar
Mochida, S. et al. Requirement for the synaptic protein interaction site for reconstitution of synaptic transmission by P/Q-type calcium channels. Proc. Natl Acad. Sci. USA 100, 2819–2824 (2003).
Article ADS CAS Google Scholar
Szabo, Z., Obermair, G. J., Cooper, C. B., Zamponi, G. W. & Flucher, B. E. Role of the synprint site in presynaptic targeting of the calcium channel CaV2.2 in hippocampal neurons. Eur. J. Neurosci. 24, 709–718 (2006).
Article Google Scholar
Bezprozvanny, I., Scheller, R. H. & Tsien, R. W. Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378, 623–626 (1995).
Article ADS CAS Google Scholar
Maximov, A., Sudhof, T. C. & Bezprozvanny, I. Association of neuronal calcium channels with modular adaptor proteins. J. Biol. Chem. 274, 24453–24456 (1999).
Article CAS Google Scholar
Kaeser, P. S. et al. RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction. Cell 144, 282–295 (2011).
Article CAS Google Scholar
Zamponi, G. W. & Currie, K. P. Regulation of Ca(V)2 calcium channels by G protein coupled receptors. Biochim. Biophys. Acta 1828, 1629–1643 (2013).
Article CAS Google Scholar
Altier, C. et al. ORL1 receptor-mediated internalization of N-type calcium channels. Nat. Neurosci. 9, 31–40 (2006).
Article CAS Google Scholar
Kisilevsky, A. E. et al. D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry. Neuron 58, 557–570 (2008).
Article CAS Google Scholar
Ramos, A. et al. The structure of the N-terminal domain of the fragile X mental retardation protein: a platform for protein-protein interaction. Structure. 14, 21–31 (2006).
Article CAS Google Scholar
Menon, R. P., Gibson, T. J. & Pastore, A. The C terminus of fragile X mental retardation protein interacts with the multi-domain Ran-binding protein in the microtubule-organising centre. J. Mol. Biol. 343, 43–53 (2004).
Article CAS Google Scholar
Lakshmana, M. K. et al. Role of RanBP9 on amyloidogenic processing of APP and synaptic protein levels in the mouse brain. FASEB J. 26, 2072–2083 (2012).
Article CAS Google Scholar
Hu, H. et al. Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release. J. Neurosci. 21, 9585–9597 (2001).
Article CAS Google Scholar
Zamponi, G. W., Lewis, R. J., Todorovic, S. M., Arneric, S. P. & Snutch, T. P. Role of voltage-gated calcium channels in ascending pain pathways. Brain Res. Rev. 60, 84–89 (2009).
Article CAS Google Scholar
Boyle, L. & Kaufmann, W. E. The behavioral phenotype of FMR1 mutations. Am. J. Med. Genet. C. Semin. Med. Genet. 154C, 469–476 (2010).
Article Google Scholar
Symons, F. J., Clark, R. D., Hatton, D. D., Skinner, M. & Bailey, D. B. Jr Self-injurious behavior in young boys with fragile X syndrome. Am. J. Med. Genet. A 118A, 115–121 (2003).
Article Google Scholar
Dolphin, A. C. & Scott, R. H. Calcium channel currents and their inhibition by (-)-baclofen in rat sensory neurones: modulation by guanine nucleotides. J. Physiol. 386, 1–17 (1987).
Article CAS Google Scholar
Berry-Kravis, E. M. et al. Effects of STX209 (arbaclofen) on neurobehavioral function in children and adults with fragile X syndrome: a randomized, controlled, phase 2 trial. Sci. Transl. Med. 4, 152ra127 (2012).
Article Google Scholar
McDonough, S. I. Calcium ion channels: challenges and successes in drug discovery. WIREs Membr. Transp. Signal. 2, 85–104 (2013).
Article CAS Google Scholar
Ferron, L. et al. 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. J. Neurosci. 28, 10604–10617 (2008).
Article CAS Google Scholar
Edbauer, D. et al. Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132. Neuron 65, 373–384 (2010).
Article CAS Google Scholar
Raghib, A. et al. Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs. J. Neurosci. 21, 8495–8504 (2001).
Article CAS Google Scholar
Kato, A. S. et al. New transmembrane AMPA receptor regulatory protein isoform, gamma-7, differentially regulates AMPA receptors. J. Neurosci. 27, 4969–4977 (2007).
Article CAS Google Scholar
Sigworth, F. J. The variance of sodium current fluctuations at the node of Ranvier. J. Physiol. 307, 97–129 (1980).
Article CAS Google Scholar
Boland, L. M., Morrill, J. A. & Bean, B. P. Omega-conotoxin block of N-type calcium channels in frog and rat sympathetic neurons. J. Neurosci. 14, 5011–5027 (1994).
Article CAS Google Scholar

Download references

Acknowledgements

We are grateful to G.J. Bassell for providing GFP-FMRP and to T.A. Ryan for providing VAMP-mCherry and vGlut1-pHluorin cDNA. We thank A. Mezghrani and A. Metz for their help with the PLA. We thank R. Willemsen for providing brain samples from wild-type and Fmr1 knockout mice. We also thank Kasumi Crews and Jackie O’Sullivan for assistance with molecular biology and K. Chaggar for technical support. We thank V. Capuano for her constructive comments on the manuscript. The monoclonal Ab 7G1-1 developed by S.T. Warren was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported by grants from the Wellcome Trust (077883/Z/) and the Medical Research Council (MR/J013285/1).

Author information

Manuela Nieto-Rostro and John S. Cassidy: These authors contributed equally to this work

Authors and Affiliations

Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK
Laurent Ferron, Manuela Nieto-Rostro, John S. Cassidy & Annette C. Dolphin

Authors

Laurent Ferron
View author publications
You can also search for this author in PubMed Google Scholar
Manuela Nieto-Rostro
View author publications
You can also search for this author in PubMed Google Scholar
John S. Cassidy
View author publications
You can also search for this author in PubMed Google Scholar
Annette C. Dolphin
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

L.F and A.C.D. designed the experiments. L.F. performed the experiments. M.N.-R. contributed to molecular biology experiments, GST pull-down assays, cell culture and data acquisition. J.S.C. contributed to molecular biology experiments. L.F. analysed the data and L.F. and A.C.D. wrote the manuscript.

Corresponding authors

Correspondence to Laurent Ferron or Annette C. Dolphin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-9 (PDF 1114 kb)

Rights and permissions

This work is licensed under a Creative Commons Attribution 3.0 Unported License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/

Reprints and permissions

About this article

Cite this article

Ferron, L., Nieto-Rostro, M., Cassidy, J. et al. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density. Nat Commun 5, 3628 (2014). https://doi.org/10.1038/ncomms4628

Download citation

Received: 29 November 2013
Accepted: 12 March 2014
Published: 07 April 2014
DOI: https://doi.org/10.1038/ncomms4628

This article is cited by

The activation of mGluR4 rescues parallel fiber synaptic transmission and LTP, motor learning and social behavior in a mouse model of Fragile X Syndrome
- Ricardo Martín
- Alberto Samuel Suárez-Pinilla
- José Sánchez-Prieto
Molecular Autism (2023)
Deletion of Fmr1 in parvalbumin-expressing neurons results in dysregulated translation and selective behavioral deficits associated with fragile X syndrome
- Magdalena Kalinowska
- Mathijs B. van der Lei
- Eric Klann
Molecular Autism (2022)
Channelopathies in fragile X syndrome
- Pan-Yue Deng
- Vitaly A. Klyachko
Nature Reviews Neuroscience (2021)
Aberrant activity of mitochondrial NCLX is linked to impaired synaptic transmission and is associated with mental retardation
- Alexandra Stavsky
- Ohad Stoler
- Israel Sekler
Communications Biology (2021)
Decreased reproducibility and abnormal experience-dependent plasticity of network dynamics in Fragile X circuits
- Helen Motanis
- Dean Buonomano
Scientific Reports (2020)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.