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Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing.

The transient receptor potential cationic channel TRPV4 contributes to different aspects of cell physiology via the generation of a Ca2+ signal and/or depolarization of the membrane potential. TRPV4 channel integrates distinct physical and chemical stimuli, including osmotic and mechanical stress, heat, acidic pH, endogenous ligands, and synthetic agonists such as 4alpha-phorbol 12,13-didecanoate (4alphaPDD). Although several regulatory sites controlling TRPV4 channel activity have been identified, very little is known about the regulation of TRPV4 expression, a situation common to other TRP channels. Here we show that TRPV4 expression is under the control of progesterone in both human airways and mammary gland epithelial cells, as well as in vascular smooth muscle cells. Exposure of human airways epithelial CFT1-LCFSN and mammary gland epithelial T47D cells to progesterone decreased TRPV4 mRNA and protein expression. Consequently, 4alphaPDD-induced cationic currents and Ca2+ signals were also diminished in progesterone-treated cells. The effect of progesterone was reverted by the progesterone receptor (PR) antagonist RU-486 or following transfection with small interference RNA (siRNA) against both PRA and PRB isoforms. Interestingly, TRPV4 expression and activity were increased in T47D mammary gland epithelial cells when PR was silenced with siRNA. Transcriptional regulation of -1.3 kB TRPV4 promoter-luciferase plasmids was also evaluated in vascular smooth muscle cells. TRPV4 promoter activity was reduced by coexpression with PR and further reduced in the presence of PG. Together, our data report the regulation of TRPV4 expression by progesterone, a process that requires a functional PR.

Calcium signaling in the brain is fundamental to the learning and memory process and there is evidence to suggest that its dysfunction is involved in the pathological pathways underlying Alzheimer’s disease (AD). Recently, the calcium hypothesis of AD has received support with the identification of the non-selective Ca2+-permeable channel CALHM1. A genetic polymorphism (p. P86L) in CALHM1 reduces plasma membrane Ca2+ permeability and is associated with an earlier age-at-onset of AD. To investigate the role of CALHM1 variants in early-onset AD (EOAD), we sequenced all CALHM1 coding regions in three independent series comprising 284 EOAD patients and 326 controls. Two missense mutations in patients (p.G330D and p.R154H) and one (p.A213T) in a control individual were identified. Calcium imaging analyses revealed that while the mutation found in a control (p.A213T) behaved as wild-type CALHM1 (CALHM1-WT), a complete abolishment of the Ca2+ influx was associated with the mutations found in EOAD patients (p.G330D and p.R154H). Notably, the previously reported p. P86L mutation was associated with an intermediate Ca2+ influx between the CALHM1-WT and the p.G330D and p.R154H mutations. Since neither expression of wild-type nor mutant CALHM1 affected amyloid ß-peptide (Aß) production or Aß-mediated cellular toxicity, we conclude that rare genetic variants in CALHM1 lead to Ca2+ dysregulation and may contribute to the risk of EOAD through a mechanism independent from the classical Aß cascade.

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We provide evidence for the association of a TRPC4 SNP with MI in population-based genetic studies. The higher Ca(2+) signals generated by TRPC4-I957V may ultimately facilitate the generation of endothelium- and nitric oxide-dependent vasorelaxation, thereby explaining its protective effect at the vasculature.

Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology.

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