Mitochondrial Ca2+-coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome

Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits, respectively, of vascular ATP-sensitive K+ (KATP) channels. In this study, we investigated changes in the vascular endothelium in mice in which Cantú syndrome-associated Kcnj8 or Abcc9 mutations were knocked in to the endogenous loci. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure or treatment with the adrenergic receptor agonist phenylephrine. We also found that either KATP GOF or acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events generated in the endothelium in response to the vasodilator agonist carbachol. Increased cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with elevated mitochondrial [Ca2+] and enhanced reactive oxygen species (ROS) and peroxynitrite levels. Scavenging intracellular or mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation, which subsequently leads to nitric oxide consumption and peroxynitrite formation, cause endothelial dysfunction in mice with Cantú syndrome

Constitutive phosphorylation of serine 29 as a critical regulator of TRPM8 channel function

Resumen del trabajo presentado al VIII Congreso Red Española de Canales Iónicos, celebrado en Alicante del 24 al 27 de mayo de 2022.The main molecular entity involved in innocuous cold detection in mammals is TRPM8. This polymodal TRP channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. Basal kinase activity phosphorylates TRPM8 and modulates its function under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. We identified four serine residues within the N-terminal domain constitutively phosphorylated in the mouse ortholog by mass spectrometry. TRPM8 function was assessed by Ca2+-imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, increased cold- and mentholevoked responses of the channel. S29A mutation is sufficient to enhance TRPM8 activity, suggesting that phosphorylation of this residue is a critical molecular determinant of this negative regulation. Biophysical and TIRF-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: an increase in the number of active channels at the plasma membrane and a shift in the voltage activation curve towards more negative potentials. Notably, basal kinase activity downregulates TRPM8 function at cold thermoreceptor neurons, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.Supported by Grants Millennium Nucleus for the Study of Pain (MiNuSPain) (RM, MP), Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD) (RM, MP), DICYT VRIDeI-USACH 022143PP (MP, RM) and by VRIDeI-USACH 021843MM (RM).Peer reviewe

TRP channels regulation of Rho GTPases in brain context and diseases

Neurological and neuropsychiatric disorders are mediated by several pathophysiological mechanisms, including developmental and degenerative abnormalities caused primarily by disturbances in cell migration, structural plasticity of the synapse, and blood-vessel barrier function. In this context, critical pathways involved in the pathogenesis of these diseases are related to structural, scaffolding, and enzymatic activity-bearing proteins, which participate in Ca2+- and Ras Homologs (Rho) GTPases-mediated signaling. Rho GTPases are GDP/GTP binding proteins that regulate the cytoskeletal structure, cellular protrusion, and migration. These proteins cycle between GTP-bound (active) and GDP-bound (inactive) states due to their intrinsic GTPase activity and their dynamic regulation by GEFs, GAPs, and GDIs. One of the most important upstream inputs that modulate Rho GTPases activity is Ca2+ signaling, positioning ion channels as pivotal molecular entities for Rho GTPases regulation. Multiple non-selective cationic channels belonging to the Transient Receptor Potential (TRP) family participate in cytoskeletal-dependent processes through Ca2+-mediated modulation of Rho GTPases. Moreover, these ion channels have a role in several neuropathological events such as neuronal cell death, brain tumor progression and strokes. Although Rho GTPases-dependent pathways have been extensively studied, how they converge with TRP channels in the development or progression of neuropathologies is poorly understood. Herein, we review recent evidence and insights that link TRP channels activity to downstream Rho GTPase signaling or modulation. Moreover, using the TRIP database, we establish associations between possible mediators of Rho GTPase signaling with TRP ion channels. As such, we propose mechanisms that might explain the TRP-dependent modulation of Rho GTPases as possible pathways participating in the emergence or maintenance of neuropathological conditions.Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDECYT 1200917 Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD) Iniciativa Cientifica Milenio of the National Agency of Research and Development (ANID), Chile Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) 21181611 21180306 21180245 21191141 MiNICAD Postdoctoral Fellowship Progra

SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a matricellular protein that modulates interactions between cells and their microenvironment. SPARC is expressed during extracellular matrix remodeling and is abundant in bone marrow and high-grade prostate cancer (PCa). In PCa, SPARC induces changes associated with epithelial–mesenchymal transition (EMT), enhancing migration and invasion and increasing the expression of EMT transcriptional factor Zinc finger E-box-binding homeobox 1 (ZEB1), but not Zinc finger protein SNAI1 (Snail) or Zinc finger protein SNAI2 (Slug). It is unknown whether the SPARC-induced downregulation of E-cadherin in PCa cells depends on ZEB1. Several integrins are mediators of SPARC effects in cancer cells. Because integrin signaling can induce EMT programs, we hypothesize that SPARC induces E-cadherin repression through the activation of integrins and ZEB1. Through stable knockdown and the overexpression of SPARC in PCa cells, we demonstrate that SPARC downregulates E-cadherin and increases vimentin, ZEB1, and integrin β3 expression. Knocking down SPARC in PCa cells decreases the tyrosine-925 phosphorylation of FAK and impairs focal adhesion formation. Blocking integrin αvβ3 and silencing ZEB1 revert both the SPARC-induced downregulation of E-cadherin and cell migration enhancement. We conclude that SPARC induces E-cadherin repression and enhances PCa cell migration through the integrin αvβ3/ZEB1 signaling pathway

Constitutive phosphorylation as a key regulator of TRPM8 channel function

In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca2+ imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.This work was supported by Grants DICYT VRIDeI-USACH 022143PP to M.P. and R.M.; FONDECYT 1161733 to R.M. and M.P.; FONDECYT 1181076 to P.O.; Centro Basal AC3E FB0008 to P.O.; VRIDeI-USACH AP-539MM to R.M.; National Research Foundation of Korea NRF-2019R1A2C2003642 to K.-S.P.; MINECO PID2019-108194RB and the Severo Ochoa Program for Centers of Excellence in R&D SEV-2017-0723 to F.V.; the Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD) to R.M. and M.P.; and the Millennium Nucleus for the Study of Pain (MiNuSPain) to R.M. and M.P. R.M. also thanks VRIDeI-USACH for postdoctoral support to B.R. (02183MM). B.R. (21161660) and B.L. (21181611) thank Comisión Nacional de Investigación Científica y Tecnológica de Chile PhD fellowships. MiNICAD is a Millennium Nucleus supported by the Millennium Science Initiative of the Ministry of Science, Technology, Knowledge and Innovation (Chile). MiNuSPain is a Millennium Nucleus supported by the Millennium Science Initiative of the Ministry of Science, Technology, Knowledge and Innovation (Chile). The CINV is a Millennium Science Institute funded by Agencia Nacional de Investigación y Desarrollo de Chile Grant ICN09_022 of the Ministry of Science, Technology, Knowledge and Innovation (Chile).Peer reviewe

KCTD5, a novel TRPM4-regulatory protein required for cell migration as a new predictor for breast cancer prognosis

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cationic channel that regulates cell migration and contractility. Increased TRPM4 expression has been related to pathologies, in which cytoskeletal rearrangement and cell migration are altered, such as metastatic cancer. Here, we identify the K+ channel tetramerization domain 5 (KCTD5) protein, a putative adaptor of cullin3 E3 ubiquitin ligase, as a novel TRPM4-interacting protein. We demonstrate that KCTD5 is a positive regulator of TRPM4 activity by enhancing its Ca2+ sensitivity. We show that through its effects on TRPM4 that KCTD5 promotes cell migration and contractility. Finally, we observed that both TRPM4 and KCTD5 expression are increased in distinct patterns in different classes of breast cancer tumor samples. Together, these data support that TRPM4 activity can be regulated through expression levels of either TRPM4 or KCTD5, not only contributing to increased understanding of the molecular mechanisms involved on the regulation of these important ion channels, but also providing information that could inform treatments based on targeting these distinct molecules that define TRPM4 activity.Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDECYT 1160518 11170291 11170223 1171155 1190806 1160900 1181263 Iniciativa Cientifica Mileni