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.

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.

Auxiliary α₂δ subunits are important proteins for trafficking of voltage-gated calcium channels (Ca_V) 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 (Ca_V2.2) 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 Ca_V channels in this component of vesicular release. We present additional evidence that Ca_V2.2 co-immunoprecipitates preferentially with cleaved wild-type α₂δ. Our data indicate that the proteolytic maturation increases the association of α₂δ−1 with Ca_V channel complex and is essential for its function on synaptic release.

Gene deletion of the voltage-gated calcium channel auxiliary subunit α ₂ δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α ₂ δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α ₂ δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α ₂ δ-1 knockout are involved in setting the duration of individual APs and their firing frequency. Our main findings are that there is reduced Ca ²⁺ entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α ₂ δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca ²⁺ buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α ₂ δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca ²⁺ -activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca ²⁺ elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α ₂ δ-1 knockout sensory neurons. This article is part of the themed issue ‘Evolution brings Ca ²⁺ and ATP together to control life and death’.

A novel missense mutation in the CACNA1A gene that encodes the pore forming α₁ subunit of the Ca_V2.1 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 Ca_V2.1 channels. Whole-cell patch clamp recordings of wild type and mutant Ca_V2.1 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 Ca_V2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in Ca_V2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.