Phosphatidylinositol-4,5-Biphosphate-Dependent Rearrangement of TRPV4 Cytosolic Tails Enables Channel Activation by Physiological Stimuli

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP$_2$), although the structural rearrangements occurring on PIP$_2$ binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP$_2$ binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence $^{121}$KRWRK$^{125}$, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP$_2$ from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP$_2$ facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP$_2$-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP$_2$ mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP$_2$ interacting site and conditions that deplete PIP$_2$ or restrict access of TRPV4 to PIP$_2$—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.Molecular and Cellular Biolog

Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21

Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2010-16725, SAF2013-46077-R, SAF2014-52228-R, BES-2011-043839), Fondo de Investigación Sanitaria (Red HERACLES RD12/0042/0014), Fundació la Marató de TV3 (20134030) and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 3150113). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset alzheimer's disease patients alter calcium homeostasis

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.This study was supported by grants from Instituto de Salud Carlos III (PI12/01311, PI10/000587, Red HERACLES RD12/0042/0014), Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain), Spanish Ministry of Economy and Competiveness (SAF2012-38140), FEDER Funds, and Generalitat de Catalunya (SGR05-266). Council of the Academy of Finland, EVO grant 5772708 of Kuopio University Hospital, the Strategic Funding of the University on Eastern Finland (UEF-Brain) (to M.H and H.S). M.A.V. is the recipient of an ICREA Academia Awar

The TRPV4 channel links calcium influx to DDX3X activity and viral infectivity

Ion channels are well placed to transduce environmental cues into signals used by cells to generate a wide range of responses, but little is known about their role in the regulation of RNA metabolism. Here we show that the TRPV4 cation channel binds the DEAD-box RNA helicase DDX3X and regulates its function. TRPV4-mediated Ca2+ influx releases DDX3X from the channel and drives DDX3X nuclear translocation, a process that involves calmodulin (CaM) and the CaM-dependent kinase II. Genetic depletion or pharmacological inhibition of TRPV4 diminishes DDX3X-dependent functions, including nuclear viral export and translation. Furthermore, TRPV4 mediates Ca2+ influx and nuclear accumulation of DDX3X in cells exposed to the Zika virus or the purified viral envelope protein. Consequently, targeting of TRPV4 reduces infectivity of dengue, hepatitis C and Zika viruses. Together, our results highlight the role of TRPV4 in the regulation of DDX3X-dependent control of RNA metabolism and viral infectivity.This work was supported by the Spanish Ministry of Economy and Competitiveness through grants SAF2015-69762R, BFU2016-80039-R, BFU2017-87843-R, an institutional “Maria de Maeztu” Programme for Units of Excellence in R&D (MDM-2014-0370) and FEDER funds; Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (PIOF-GA-2009-237120) and the Generalitat de Catalunya research program (AGAUR, 2014-SGR-1628 and FI-2013FIB00251)

The mechanosensitive Piezo1 channel controls endosome trafficking for an efficient cytokinetic abscission

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.This study was supported by the Spanish Ministry of Science, Education, and Universities through grants RTI2018-099718-B-100 to M.A.V. and PGC2018-095663-B100 to C.P., an institutional “María de Maeztu” Programme for Units of Excellence in R&D, and FEDER funds. C.P. is the recipient of an ICREA Academia award

Methylglyoxal reduces mitochondrial potential and activates Bax and caspase-3 in neurons: implications for alzheimer's disease

Alzheimer's disease (AD) is characterized by the oxidative stress generated from amyloid β-peptide (Aβ) aggregates. It produces protein nitrotyrosination, after the reaction with nitric oxide to form peroxynitrite, being triosephosphate isomerase (TPI) one of the most affected proteins. TPI is a glycolytic enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Methylglyoxal (MG) is a by-product of TPI activity whose production is triggered when TPI is nitrotyrosinated. MG is harmful to cells because it glycates proteins. Here we found protein glycation when human neuroblastoma cells were treated with Aβ. Moreover glycation was also observed when neuroblastoma cells overexpressed mutated TPI where Tyr165 or Tyr209, the two tyrosines close to the catalytic center, were changed by Phe in order to mimic the effect of nitrotyrosination. The pathological relevance of these findings was studied by challenging cells with Aβ oligomers and MG. A significant decrease in mitochondrial transmembrane potential, one of the first apoptotic events, was obtained. Therefore, increasing concentrations of MG were assayed searching for MG effect in neuronal apoptosis. We found a decrease of the protective Bcl2 and an increase of the proapoptotic caspase-3 and Bax levels. Our results suggest that MG is triggering apoptosis in neurons and it would play a key role in AD neurodegeneration.This work was supported by the Plan Estatal de I+D+i 2013-2016 and the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Grants PI13/00408, and Red HERACLES RD12/0042/0014) and FEDER Funds; the virtual physiological human (VPH) NoE (FP7-ICT-2007-2-223920), the Spanish Ministry of Science and Innovation (CTQ2008-00755; BFU2006-28430-E/BMC and RETIC COMBIOMED RD07/0067/0001); Generalitat de Catalunya (AGAUR BE-2 10240); and La Marató de TV3 (Nº 100310)

LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions

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)

Methylglyoxal reduces mitochondrial potential and activates Bax and caspase-3 in neurons: implications for alzheimer's disease

Alzheimer's disease (AD) is characterized by the oxidative stress generated from amyloid β-peptide (Aβ) aggregates. It produces protein nitrotyrosination, after the reaction with nitric oxide to form peroxynitrite, being triosephosphate isomerase (TPI) one of the most affected proteins. TPI is a glycolytic enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Methylglyoxal (MG) is a by-product of TPI activity whose production is triggered when TPI is nitrotyrosinated. MG is harmful to cells because it glycates proteins. Here we found protein glycation when human neuroblastoma cells were treated with Aβ. Moreover glycation was also observed when neuroblastoma cells overexpressed mutated TPI where Tyr165 or Tyr209, the two tyrosines close to the catalytic center, were changed by Phe in order to mimic the effect of nitrotyrosination. The pathological relevance of these findings was studied by challenging cells with Aβ oligomers and MG. A significant decrease in mitochondrial transmembrane potential, one of the first apoptotic events, was obtained. Therefore, increasing concentrations of MG were assayed searching for MG effect in neuronal apoptosis. We found a decrease of the protective Bcl2 and an increase of the proapoptotic caspase-3 and Bax levels. Our results suggest that MG is triggering apoptosis in neurons and it would play a key role in AD neurodegeneration.This work was supported by the Plan Estatal de I+D+i 2013-2016 and the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Grants PI13/00408, and Red HERACLES RD12/0042/0014) and FEDER Funds; the virtual physiological human (VPH) NoE (FP7-ICT-2007-2-223920), the Spanish Ministry of Science and Innovation (CTQ2008-00755; BFU2006-28430-E/BMC and RETIC COMBIOMED RD07/0067/0001); Generalitat de Catalunya (AGAUR BE-2 10240); and La Marató de TV3 (Nº 100310)

Structural determinants of 5′,6′-epoxyeicosatrienoic acid binding to and activation of TRPV4 channel

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 fellowshi