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Data de publicació electrònica: 02-11-2022 Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology. This work was supported in part by R01 CA257647 (to K.K. and D.M.G.), R01 GM134542 (to S.X.S. and K.K.), NSF 2045715 (to Y.L.), R01 AR071976 (to C.-M.F. and J.T.), R01 AR072644 (to C.-M.F. and J.T.) and R01 CA054358 (to A.P.F.), the Spanish Ministry of Science, Education and Universities through grants RTI2018 099718-B-100 (to M.A.V.) and an institutional “Maria de Maeztu” Programme for Units of Excellence in R&D and FEDER funds (to M.A.V.), and postdoctoral fellowships from the Fonds de recherche du Quebec—Nature et technologies and the Natural Sciences and Engineering Research Council of Canada (to A.K.). The opinions, findings and conclusions, or recommendations expressed are those of the authors and do not necessarily reflect the views of any of the funding agencies.

Description, Piezo1 transduces mechanical forces at the intercellular bridge to coordinate the machinery necessary to split dividing cells., Mechanical forces are exerted throughout cytokinesis, the final step of cell division. Yet, how forces are transduced and affect the signaling dynamics of cytokinetic proteins remains poorly characterized. We now show that the mechanosensitive Piezo1 channel is activated at the intercellular bridge (ICB) connecting daughter cells to regulate abscission. Inhibition of Piezo1 caused multinucleation both in vitro and in vivo. Piezo1 positioning at the ICB during cytokinesis depends on Pacsin3. Pharmacological and genetic inhibition of Piezo1 or Pacsin3 resulted in mislocation of Rab11-family-interacting protein 3 (Rab11-FIP3) endosomes, apoptosis-linked gene 2-interacting protein X (ALIX), and endosomal sorting complex required for transport III (ESCRT-III). Furthermore, we identified FIP3 as the link between Piezo1-generated Ca2+ signals and ALIX delivery to the ICB, where ALIX recruits the ESCRT-III component charged multivesicular body protein 4B, which promotes abscission. These results provide a different view of how mechanical forces participate in cytokinesis and identify Piezo1 as a key modulator of endosome trafficking.

HL-1 cells; L-type calcium channels; Calcium regulation Células HL-1; Canales de calcio tipo L; Regulación del calcio Cel·lules HL-1; Canals de calci tipus L; Regulació del calci MicroRNAs (miRNAs) participate in atrial remodeling and atrial fibrillation (AF) promotion. We determined the circulating miRNA profile in patients with AF and heart failure with reduced ejection fraction (HFrEF), and its potential role in promoting the arrhythmia. In plasma of 98 patients with HFrEF (49 with AF and 49 in sinus rhythm, SR), differential miRNA expression was determined by high-throughput microarray analysis followed by replication of selected candidates. Validated miRNAs were determined in human atrial samples, and potential arrhythmogenic mechanisms studied in HL-1 cells. Circulating miR-199a-5p and miR-22-5p were significantly increased in HFrEF patients with AF versus those with HFrEF in SR. Both miRNAs, but particularly miR-199a-5p, were increased in atrial samples of patients with AF. Overexpression of both miRNAs in HL-1 cells resulted in decreased protein levels of L-type Ca2+ channel, NCX and connexin-40, leading to lower basal intracellular Ca2+ levels, fewer inward currents, a moderate reduction in Ca2+ buffering post-caffeine exposure, and a deficient cell-to-cell communication. In conclusion, circulating miR-199a-5p and miR-22-5p are higher in HFrEF patients with AF, with similar findings in human atrial samples of AF patients. Cells exposed to both miRNAs exhibited altered Ca2+ handling and defective cell-to-cell communication, both findings being potential arrhythmogenic mechanisms. This work was funded by the following grants, awarded to B.B.: Sociedad Española de Cardiología, Sección de Arritmias y Electrofisiología 2012; Sociedad Española de Cardiología, Sección de Insuficiencia Cardíaca 2013; Fondo Investigación Sanitaria (FIS)—Instituto Carlos III 2013 (PI13/01830); and Societat Catalana de Cardiologia 2016. Awarded to K.W.A-A. and S.R.: British Heart Foundation (BHF) Intermediate Research Fellowship. Awarded to J.M.F.F.: grant from the Spanish Ministry of Science and Innovation (RTI2018-094809-B-I00). “María de Maeztu” Programme for Units of Excellence in R&D to the Departament de Ciències Experimentals i de la Salut (MDM-2014-0370) and FEDER (Fondo Europeo de Desarrollo Regional) also contributed to this work.

Fluid shear sensing via TRPM7 limits tumor cell intravasation by activating RhoA/myosin and calmodulin/IQGAP1/Cdc42 pathways., Tumor cell intravasation preferentially occurs in regions of low fluid shear because high shear is detrimental to tumor cells. Here, we describe a molecular mechanism by which cells avoid high shear during intravasation. The transition from migration to intravasation was modeled using a microfluidic device where cells migrating inside longitudinal tissue-like microchannels encounter an orthogonal channel in which fluid flow induces physiological shear stresses. This approach was complemented with intravital microscopy, patch-clamp, and signal transduction imaging techniques. Fluid shear–induced activation of the transient receptor potential melastatin 7 (TRPM7) channel promotes extracellular calcium influx, which then activates RhoA/myosin-II and calmodulin/IQGAP1/Cdc42 pathways to coordinate reversal of migration direction, thereby avoiding shear stress. Cells displaying higher shear sensitivity due to higher TRPM7 activity levels intravasate less efficiently and establish less invasive metastatic lesions. This study provides a mechanistic interpretation for the role of shear stress and its sensor, TRPM7, in tumor cell intravasation.

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl- efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+/K+/2Cl- cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions. This work was supported by grants from the Ministry of Science, Innovation, and Universities (PGC2018-094136-B-I00 to F.P.; BFU2017-85152-P and Fondo Europeo de Desarrollo Regional [FEDER] to E.d.N.; RTI2018-099718-B-I00 and FEDER to M.A.V.), the Catalan Government (2017 SGR 799), the Fundación Botín, and the Banco Santander through its Santander Universities Global Division to F.P. We gratefully acknowledge institutional funding from the Ministry of Science, Innovation and Universities through the Centres of Excellence Severo Ochoa Award and from the Centres de Recerca de Catalunya (CERCA) Programme of the Catalan Government and the Unidad de Excelencia María de Maeztu, funded by the Agencia Estatal de Investigación (AEI) (CEX2018-000792-M). F.P. and E.d.N. are recipients of an Institució Catalana de Recerca i Estudis Avançats (ICREA) Acadèmia award (Generalitat de Catalunya).

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

Aims: Single nucleotide polymorphisms on chromosome 4q25 have been associated with risk of atrial fibrillation (AF) but the exiguous knowledge of the mechanistic links between these risk variants and underlying electrophysiological alterations hampers their clinical utility. Here, we here tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homeostasis that predispose to spontaneous electrical activity. Methods and results: Western blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homeostasis in human atrial myocytes. Our findings revealed that the rs13143308T variant was more frequent in patients with AF and that myocytes from carriers of this variant had a significantly higher density of calcium sparks (14.1±4.5 vs. 3.1±1.3 events/min, p¿=¿0.02), frequency of transient inward (ITI) currents (1.33±0.24 vs. 0.26±0.09 events/min, p¿, This work was supported by multi-centric grants from Centro Nacional de Investigaciones Cardiovasculares [CNIC-2009-08 to L.H.-M. and D.F.]; a grant from Fundacio´ Marato´ TV3 [2015-20-30 to L.H.-M.]; and grants from the Spanish Ministry of Economy and Competition [SAF2014-58286-C2-1-R to L.H.-M.] and [DPI2013-44584-R to R.B.]; and from the Spanish Ministry of Health and Consume, Instituto de Salud Carlos III, Red de Investigacio´n Cardiovascular [RD12/0042/0002] and CIBERCV to J.C., and from Fondo Europeo de Desarrollo Regional (FEDER).

SUMMARY Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca2+ elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments., In Brief Hung et al. demonstrate that a Piezo1-dependent intracellular calcium increase negatively regulates protein kinase A (PKA) as cells transit from unconfined to confined spaces. The Piezo1/PKA and myosin II signaling modules constitute two confinement-sensing mechanisms. This study provides a paradigm by which signaling enables cells to sense and adapt to different microenvironments.

Aims Atrial fibrillation (AF) is the most common type of arrhythmia in humans, yet the genetic cause of AF remains elusive. Genome-wide association studies (GWASs) have reported risk variants in four distinct genetic loci, and more recently, a meta-GWAS has further implicated six new loci in AF. However, the functional role of these AF GWAS-related genes in AF and their inter-relationship remain elusive. Methods and results To get further insights into the molecular mechanisms driven by Pitx2, calcium handling and novel AFGWAS-associated gene expression were analysed in two distinct Pitx2 loss-of-function models with distinct basal electrophysiological defects; a novel Pitx2 conditional mouse line, Sox2CrePitx2, and our previously reported atrial-specific NppaCrePitx2 line. Molecular analyses of the left atrial appendage in NppaCrePitx2(+/-) and NppaCrePitx2(-/-) adult mice demonstrate that AF GWAS-associated genes such as Zfhx3, Kcnn3, and Wnt8a are severely impaired but not Cav1, Synpo2l, nor Prrx1. In addition, multiple calcium-handling genes such as Atp2a2, Casq2, and Plb are severely altered in atrial-specific NppaCrePitx2 mice in a dose-dependent manner. Functional assessment of calcium homeostasis further underscores these findings. In addition, multiple AF-related microRNAs are also impaired. In vitro over-expression of Wnt8, but not Zfhx3, impairs calcium handling and modulates microRNA expression signature identified in Pitx2 loss-of-function models. ConclusionOur data demonstrate a dose-dependent relation between Pitx2 expression and the expression of AF susceptibility genes, calcium handling, and microRNAs and identify a complex regulatory network orchestrated by Pitx2 with large impact on atrial arrhythmogenesis susceptibility.

Modulation of CaV2.1 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 CaV2.1 pore‐forming α 1A 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 CaV2.1 also promotes channel inactivation. This process seems to involve Ca2+‐calmodulin interaction with two adjacent sites in the α 1A 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 β subunits to the α interaction domain (AID) at the first intracellular loop (LI‐II) of α 1A determines the degree of channel inactivation by both voltage and Ca2+. Here, we explore the cross talk between β subunits, Ca2+, and syntaxin‐1A‐modulated CaV2.1 inactivation, highlighting the α 1A domains involved in such process. β 3‐containing CaV2.1 channels show syntaxin‐1A‐modulated but no Ca2+‐dependent steady‐state inactivation. Conversely, β 2a‐containing CaV2.1 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 β 3‐containing CaV2.1 channels. Mutation of the IQ‐like motif, unlike CBD deletion, abolishes Ca2+‐dependent inactivation and confers modulation by syntaxin‐1A in β 2a‐containing CaV2.1 channels. Altogether, these results suggest that LI‐II structural modifications determine the regulation of CaV2.1 steady‐state inactivation either by Ca2+ or by SNAREs but not by both.

TRPV4 cation channel activation by cytochrome P450-mediated derivatives of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs), constitute a major mechanisms of endothelium-derived vasodilatation. Besides, TRPV4 mechano/osmosensitivity depends on phospholipase A2 (PLA2) activation and subsequent production of AA and EETs. However, the lack of evidence for a direct interaction of EETs with TRPV4 together with claims of EET-independent mechanical activation of TRPV4 has cast doubts on the validity of this mechanism. We now report: 1) The identification of an EET-binding pocket that specifically mediates TRPV4 activation by 5′,6′-EET, AA and hypotonic cell swelling, thereby suggesting that all these stimuli shared a common structural target within the TRPV4 channel; and 2) A structural insight into the gating of TRPV4 by a natural agonist (5′,6′-EET) in which K535 plays a crucial role, as mutant TRPV4-K535A losses binding of and gating by EET, without affecting GSK1016790A, 4α-phorbol 12,13-didecanoate and heat mediated channel activation. Together, our data demonstrates that the mechano- and osmotransducing messenger EET gates TRPV4 by a direct action on a site formed by residues from the S2-S3 linker, S4 and S4-S5 linker. The research is supported by awards from the Spanish Ministry of Economy and Competitiveness (Grants SAF2015-69762-R to M.A.V. and J.M.F.-F., and MDM-2014-0370 through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”), and FEDER Funds (Fondo Europeo de Desarrollo Regional). M.I.-S. holds a “Juan de la Cierva-Formación” Fellowship funded by the Spanish Ministry of Economy and Competitiveness. FGN acknowledge the support of FONDECYT Grant 1170733 and The Centro Interdisciplinario de Neurociencia de Valparaíso (CINV) is a Millennium Institute supported by the Millennium Scientific Initiative of the Ministerio de Economía, Fomento y Turismo. R.V.S. is funded by CONICYT PCHA/Doctorado Nacional 2013-21130631 fellowship

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 β 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 β subunits, Ca2+ , and syntaxin-1A-modulated CaV 2.1 inactivation, highlighting the α1A domains involved in such process. β3 -containing CaV 2.1 channels show syntaxin-1A-modulated but no Ca2+ -dependent steady-state inactivation. Conversely, β2a -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 β3 -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 β2a -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. This work was supported by the Spanish Ministry of Economy and Competitiveness (Grants SAF2012‐31089 and SAF2015‐69762‐R to JMF‐F, and Grant MDM‐2014‐0370 through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”), and FEDER Funds.

Familial hemiplegic migraine (FHM)-causing mutations in the gene encoding the P/Q Ca 2+ channel α 1A subunit ( CACNA1A ) locate to the pore and voltage sensor regions and normally involve gain-of-channel function. We now report on a mutation identified in the first intracellular loop of CACNA1A (α 1A(A454T) ) that does not cause FHM but is associated with the absence of sensorimotor symptoms in a migraine with aura pedigree. α 1A(A454T) channels showed weakened regulation of voltage-dependent steady-state inactivation by Ca V β subunits. More interestingy, A454T mutation suppressed P/Q channel modulation by syntaxin 1A or SNAP-25 and decreased exocytosis. Our findings reveal the importance of I-II loop structural integrity in the functional interaction between P/Q channel and proteins of the vesicle-docking/fusion machinery, and that genetic variation in CACNA1A may be not only a cause but also a modifier of migraine phenotype.

Migraine is a common neurological disorder with a complex inheritance pattern. Mutations in genes encoding proteins that are involved in ion transport across the neuronal membrane have been linked to rare monogenic variants of migraine. These or other related genes and proteins are also candidates to be involved in the inherited predisposition to the more common forms of migraine without aura (MO) or migraine with aura (MA). One of these proteins, syntaxin 1A, encoded by the STX1A gene, is a key molecule in ion channel regulation and synaptic exocytosis. We assessed the contribution of STX1A to migraine by analyzing three SNPs that cover the entire gene (rs6951030–rs941298–rs4363087), in a case–control association study in 210 migraine patients (102 MO, 86 MA, 22 hemiplegic migraine) and 210 sex-matched unrelated controls. The single-marker analysis revealed significant differences in both allele frequencies (P = 0.0087, OR = 1.48) and genotype distributions (P = 0.0133) of the rs941298 SNP between migraineurs and controls, with an overrepresentation of T-allele carriers in the migraine sample (OR = 1.78). We subsequently performed a haplotype-based analysis and observed evidence of an overrepresentation of the A–T–G (rs6951030–rs941298–rs4363087) allelic combination in migraine patients and an increased frequency of carriers of this risk haplotype (P = 0.008, OR = 1.71). These differences remained significant when patients were subdivided into MO and MA. When the control series was enlarged for rs941298, we confirmed the association only with the whole migraine group.

We report a patient with typical features of episodic ataxia type 2 (EA2) but with onset in the sixth decade and associated interictal hand dystonia. He was found to bear the novel heterozygous missense mutation p.Gly638Asp (c.1913G > A) in the CACNA1A gene. Functional analysis of the mutation on P/Q channels expressed in HEK 293 cells revealed a reduction of Ca2+ current densities, a left-shift in the apparent reversal potential, the slowing of inactivation kinetics and the increase in the rate of current recovery from inactivation. These results are consistent with a decrease in Ca2+ permeability through mutant P/Q channels. To our knowledge, this is just the second patient with late onset EA2 linked to a CACNA1A mutation and the first to carry a loss-of-function missense mutation., This study was supported by the Spanish Ministry of Education and Science (SAF2003-04704, SAF2006-13893-C02-01, SAF2006-13893-C02-02, SAF2006-4973), Fondo de Investigación Sanitaria (red HERACLES), Fundació La Marató de TV3 (061330, 061331) and Agència de Gestió d'Ajuts Universitaris i de Recerca-AGAUR (2005SGR00848 and 2005SGR00266). RC was funded by the Institut de Recerca Vall d'Hebron, MR and EC-L are recipients of a Juan de la Cierva and a Marató de TV3 contracts, respectively, and SAS is a recipient of a FPI scholarship of the Spanish Ministry of Education and Science. JMF-F is a Ramón y Cajal Fellow. MAV is an ICREA Academia Fellow.

Mutations in the gene encoding the pore-forming alpha(1A) subunit of P/Q Ca(2+) channels (CACNA1A) are linked to familial hemiplegic migraine. CACNA1A Y1245C is the first missense mutation described in a subject affected with childhood periodic syndromes that evolved into hemiplegic migraine. Y1245C is also the first amino acid change described in any S1 segment of CACNA1A in a hemiplegic migraine background. We found that Y1245C induced a 9-mV left shift in the current-voltage activation curve, accelerated activation kinetics, and slowed deactivation kinetics within a wide range of voltage depolarizations. Y1245C also left-shifted the voltage-dependent steady-state inactivation with a significant increase in steepness, suggesting a direct effect on the P/Q channel voltage sensor. Moreover, Y1245C reduced Gbetagamma subunits-dependent channel inhibition probably by favoring Gbetagamma dissociation from the channel; an effect also observed using action-potential-like waveforms of different durations. The formation of a new disulfide bridge between cysteines may contribute to the Y1245C effects on activation and Gbetagamma inhibition of the channel, as they were significantly reversed by the sulphydryl-reducing agent dithiothreitol. Together, our data suggest that Y1245C alters the structure of the alpha(1A) voltage sensor producing an overall gain of channel function that may explain the observed clinical phenotypes.

Oxidative stress (OS) underlies neuronal dysfunction in many neurodegenerative disorders. Regulator of Calcineurin 1 (RCAN1 or DSCR1) is a dose-sensitive gene whose overexpression has been linked to Down syndrome (DS) and Alzheimer's disease (AD) neuropathology and to the response of cells to stress stimuli. Here, we show that RCAN1 mRNA and protein expression are sensitive to OS in primary neurons, and we evaluate the involvement of RCAN1 dosage in neuronal death induced by OS. We find that Rcan1(-/-) neurons display an increased resistance to damage by H(2)O(2), which can be reverted by RCAN1 overexpression or by exogenous inhibitors of calcineurin. Although increased intracellular Ca(2+) concentration is an important factor in OS-mediated cell death, our results show that Ca(2+) loading after exposure to H(2)O(2) was similar in Rcan1(+/+) and Rcan1(-/-) neurons. Our data further suggest that CaN and NFAT signaling protect against OS in both Rcan1(+/+) and Rcan1(-/-) neurons. To explain the observed differential vulnerability, we therefore propose a mechanism downstream of H(2)O(2)-mediated Ca(2+) entry, involving calcineurin-NFAT signaling. These findings highlight the importance of RCAN1 gene dosage in the modulation of cell survival and death pathways and suggest that changes in the amount of RCAN1 could represent an important mechanism for regulating susceptibility to neurodegeneration, especially in DS and AD.

The transcription factor Pitx2 has been proposed as a molecular link between single nucleotide polymorphisms on chromosome 4q25 and increased risk of atrial fibrillation in carriers of the risk variant. Since atrial fibrillation has been associated with calcium handling disturbances in isolated atrial myocytes, we here tested the hypothesis that Pitx2 insufficiency alters the calcium homeostasis in atrial myocytes.To test this hypothesis, we used right atrial myocytes from a transgenic mouse model with inducible atrial specific Pitx2 deletion. Spontaneous calcium release was detected with confocal calcium imaging and resulting ion currents or membrane depolarizations were measured with patch-clamp technique in myocytes from wild-type (Pitx2+/+) and heterozygous Pitx2+/- mice.Calcium imaging revealed that the frequency of calcium sparks (2.1±0.7 vs 0.2±0.1 events/cell/s, p

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.

International audience; BACKGROUND & AIMS: Acinar cells constitute 90% of the pancreas epithelium, are polarized, and secrete digestive enzymes. These cells play a crucial role in pancreatitis and pancreatic cancer. However, there are limited models to study normal acinar cell differentiation in vitro. The aim of this work was to generate and characterize purified populations of pancreatic acinar cells from embryonic stem (ES) cells. METHODS: Reporter ES cells (Ela-pur) were generated that stably expressed both beta-galactosidase and puromycin resistance genes under the control of the elastase I promoter. Directed differentiation was achieved by incubation with conditioned media of cultured fetal pancreatic rudiments and adenoviral-mediated co-expression of p48/Ptf1a and Mist1, 2 basic helix-loop-helix transcription factors crucial for normal pancreatic acinar development and differentiation. RESULTS: Selected cells expressed multiple markers of acinar cells, including digestive enzymes and proteins of the secretory pathway, indicating activation of a coordinated differentiation program. The genes coding for digestive enzymes were not regulated as a single module, thus recapitulating what occurs during in vivo pancreatic development. The generated cells displayed transient agonist-induced Ca(2+) mobilization and showed a typical response to physiologic concentrations of secretagogues, including enzyme synthesis and secretion. Importantly, these effects did not imply the acquisition of a mixed acinar-ductal phenotype. CONCLUSIONS: These studies allow the generation of almost pure acinar-like cells from ES cells, providing a normal cell-based model for the study of the acinar differentiation program in vitro.

Purpose: Atrial fibrillation has been associated with risk variants at chromosome 4q25 that presumably modulate the expression or activity of the transcription factor pitx2. Moreover, pitx2 expression is much higher in left than right atrium, and we here tested the hypothesis that pitx2 modulates cardiomyocyte features associated to atrial fibrillation such altered cell size and spontaneous calcium release differentially in right and left atrial myocytes. Methods: Perforated patch-clamp technique was applied to left and right atrial myocytes from mice with or without atrial chamber-specific deletion of pitx-2 to measure cell capacitance. Current-clamp was used to record the frequency of spontaneous action potentials at different membrane potentials by clamping the holding current at different values. Results: Comparison of the cell surface area, estimated from the cell capacitance, revealed that left atrial myocytes had a larger cell surface than right atrial myocytes (62±5 vs. 42±3 pC/pF, p=0.01) in wild type mice. Moreover, myocytes from pitx2+/- mice had a larger cell capacitance than those from wild type mice (80±6 vs. 62±5 pC/pF, p=0.06 for left atria and 69±7 vs. 42±3 pC/pF, p=0.03 for right atria). In wild type mice, spontaneous action potentials were rare in both left and right atrial myocytes and were not observed at a normal resting membrane potentials (-80 mV). In pitx2+/- mice, spontaneous action potentials were rarely observed in left atrial myocytes, even when the resting membrane potential was increased to -72±1 mV (0.05±0.05 events/min) or to -63±1 mV (0.3±0.3 events/min). By contrast, spontaneous action potentials were observed even at -77.8±0.4 mV in right atrial myocytes (0.8±0.6 events/min) and their frequency increased dramatically to 2.5±1.5 events/min at -72±1 mV and 13.8±7.92 events/min at -63±1 mV. Conclusions: Loss of pitx2 expression results in myocytes with features associated to atrial fibrillation such as increased myocyte surface and spontaneous action potentials. Moreover, the frequency of spontaneous action potentials is selectively increased in right atrial myocytes from pitx2+/- mice, suggesting that they are the key source of arrhytmogenic action potentials.

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 three genes (CACNA1A, ATP1A2 and SCN1A) 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.

TRPM7 senses hydraulic pressure and modulates cortical actomyosin to guide migrating cells towards the path of lower resistance., How cells sense hydraulic pressure and make directional choices in confinement remains elusive. Using trifurcating Ψ-like microchannels of different hydraulic resistances and cross-sectional areas, we discovered that the TRPM7 ion channel is the critical mechanosensor, which directs decision-making of blebbing cells toward channels of lower hydraulic resistance irrespective of their cross-sectional areas. Hydraulic pressure–mediated TRPM7 activation triggers calcium influx and supports a thicker cortical actin meshwork containing an elevated density of myosin-IIA. Cortical actomyosin shields cells against external forces and preferentially directs cell entrance in low resistance channels. Inhibition of TRPM7 function or actomyosin contractility renders cells unable to sense different resistances and alters the decision-making pattern to cross-sectional area–based partition. Cell distribution in microchannels is captured by a mathematical model based on the maximum entropy principle using cortical actin as a key variable. This study demonstrates the unique role of TRPM7 in controlling decision-making and navigating migration in complex microenvironments.

Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase A(2) (PLA(2)) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA(2)-EET as the primary pathway to activate TRPV4. Under conditions of low PLA(2) activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)-inositol trisphosphate (IP(3)) signaling to support TRPV4 gating. IP(3), without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing TRPV4, an effect inhibited by the IP(3) receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP(3) receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular store Ca(2+) channels required to initiate and maintain the oscillatory Ca(2+) signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA(2) activation. This work was supported by grants from the Spanish Ministries of Education and Science (SAF2006-04973 and SAF2006-13893-C02-02), and Health (Fondo de Investigación Sanitaria, Red HERACLES RD06/0009), the Generalitat de Catalunya (SGR05-266), and Fundació la Marató de TV3 (061331). J.M. Fernández-Fernández is a Ramón y Cajal Fellow.