Cross talk between ß subunits, intracellular Ca2+ signaling, and SNAREs in the modulation of CaV 2.1 channel steady-state inactivation.

Modulation of CaV 2.1 channel activity plays a key role in interneuronal communication and synaptic plasticity. SNAREs interact with a specific synprint site at the second intracellular loop (LII-III) of the CaV 2.1 pore-forming α1A subunit to optimize neurotransmitter release from presynaptic terminals by allowing secretory vesicles docking near the Ca2+ entry pathway, and by modulating the voltage dependence of channel steady-state inactivation. Ca2+ influx through CaV 2.1 also promotes channel inactivation. This process seems to involve Ca2+ -calmodulin interaction with two adjacent sites in the α1A carboxyl tail (C-tail) (the IQ-like motif and the Calmodulin-Binding Domain (CBD) site), and contributes to long-term potentiation and spatial learning and memory. Besides, binding of regulatory ß subunits to the α interaction domain (AID) at the first intracellular loop (LI-II) of α1A determines the degree of channel inactivation by both voltage and Ca2+ . Here, we explore the cross talk between ß subunits, Ca2+ , and syntaxin-1A-modulated CaV 2.1 inactivation, highlighting the α1A domains involved in such process. ß3 -containing CaV 2.1 channels show syntaxin-1A-modulated but no Ca2+ -dependent steady-state inactivation. Conversely, ß2a -containing CaV 2.1 channels show Ca2+ -dependent but not syntaxin-1A-modulated steady-state inactivation. A LI-II deletion confers Ca2+ -dependent inactivation and prevents modulation by syntaxin-1A in ß3 -containing CaV 2.1 channels. Mutation of the IQ-like motif, unlike CBD deletion, abolishes Ca2+ -dependent inactivation and confers modulation by syntaxin-1A in ß2a -containing CaV 2.1 channels. Altogether, these results suggest that LI-II structural modifications determine the regulation of CaV 2.1 steady-state inactivation either by Ca2+ or by SNAREs but not by both.

A loss-of-function CACNA1A mutation causing benign paroxysmal torticollis of infancy.

Benign paroxysmal torticollis of infancy (BPTI) is a rare paroxysmal disorder characterized by recurrent episodes of head tilt and accompanying general symptoms which remit spontaneously. The rare association with gain-of-function CACNA1A mutations, similar to hemiplegic migraine, has been reported. We report here two new BPTI patients from the same family carrying a heterozygous mutation in the CACNA1A gene leading to the change p.Glu533Lys. Functional analysis revealed that this mutation induces a loss of channel function due to impaired gating by voltage and much lower current density. Our data suggest that BPTI, a periodic syndrome commonly considered a migraine precursor, constitutes an age-specific manifestation of defective neuronal calcium channel activity.

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies.

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.

A gain-of-function SNP in TRPC4 cation channel protects against myocardial infarction.

AIMS: The TRPC4 non-selective cation channel is widely expressed in the endothelium, where it generates Ca(2+) signals that participate in the endothelium-mediated vasodilatory response. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC4 gene that are associated with myocardial infarction (MI). METHODS AND RESULTS: Our candidate-gene association studies identified a missense SNP (TRPC4-I957V) associated with a reduced risk of MI in diabetic patients [odds ratio (OR) = 0.61; confidence interval (CI), 0.40-0.95, P= 0.02]. TRPC4 was also associated with MI in the Wellcome Trust Case-Control Consortium's genome-wide data: an intronic SNP (rs7319926) within the same linkage disequilibrium block as TRPC4-I957V showed an OR of 0.86 (CI, 0.81-0.94; P =10(-4)). Functional studies of the missense SNP were carried out in HEK293 and CHO cells expressing wild-type or mutant channels. Patch-clamp studies and measurement of intracellular [Ca(2+)] in response to muscarinic agonists and direct G-protein activation showed increased channel activity in TRPC4-I957V-transfected cells compared with TRPC4-WT. Site-directed mutagenesis and molecular modelling of TRPC4-I957V suggested that the gain of function was due to the presence of a less bulky Val-957. This permits a firmer interaction between the TRPC4 and the catalytic site of the tyrosine kinase that phosphorylates TRPC4 at Tyr-959 and facilitates channel insertion into the plasma membrane. CONCLUSION: 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.

The progesterone receptor regulates the expression of TRPV4 channel.

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.