Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

Insights into the pathogenesis of migraine with aura may be gained from a study of human CaV2.1 channels containing mutations linked to familial hemiplegic migraine (FHM). Here, we extend the previous single-channel analysis to human CaV2.1 channels containing mutation V1457L. This mutation increased the channel open probability by shifting its activation to more negative voltages and reduced both the unitary conductance and the density of functional channels in the membrane. To investigate the possibility of changes in Ca V2.1 function common to all FHM mutations, we calculated the product of single-channel current and open probability as a measure of Ca 2؉ influx through single CaV2.1 channels. All five FHM mutants analyzed showed a single-channel Ca 2؉ influx larger than wild type in a broad voltage range around the threshold of activation. We also expressed the FHM mutants in cerebellar granule cells from CaV2.1␣1 ؊/؊ mice rather than HEK293 cells. The FHM mutations invariably led to a decrease of the maximal CaV2.1 current density in neurons. Current densities were similar to wild type at lower voltages because of the negatively shifted activation of FHM mutants. Our data show that mutational changes of functional channel densities can be different in different cell types, and they uncover two functional effects common to all FHM mutations analyzed: increase of single-channel Ca 2؉ influx and decrease of maximal CaV2.1 current density in neurons. We discuss the relevance of these findings for the pathogenesis of migraine with aura.

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw’s nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients’ quality of life.

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In experimental and clinical pain studies, the sex of subjects was rarely taken into account, even if nociceptive inputs appear to be processed and modulated by partially distinct neural mechanisms in each sex. In this study we analysed, in male and female mice, behavioural and neuronal responses in developing, maintaining, and recovering from neuropathic pain. Experiments were carried out in adult CD1 mice by using Chronic Constriction Injury (CCI) as neuropathic pain model. We investigated the temporal trend of mechanical nociceptive threshold together with functional recovery of the injured paw, and the immunofluorescence staining of proteins associated with nerve injury and repair and with spinal gliosis, 7 and 121days after CCI. A proteomic analysis on proteins extracted from sciatic nerves was also performed. Male mice showed a gradual decrease of CCI-induced allodynia, the complete recovery occurring 81days after the sciatic nerve ligation. On the contrary, in female mice, allodynia was still present 121days after CCI. Sex-dependent differences also resulted from immunofluorescence experiments: in sciatic nerve, the expression of P0 and Neu200 is greater in neuropathic males than in neuropathic females, suggesting faster nerve regeneration. Proteomic analysis confirmed sex-related differences of proteins associated with nerve regenerative processes. In addition, the reactive gliosis induced by CCI at day 7, as revealed by colocalization of glial fibrillary acidic protein (astrocytes) and CD11b (microglia) with phosphorylated p38, disappeared 121 days after CCI in male but not in female mice. These results may have important therapeutic implications for the treatment of neuropathic pain.

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