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      Obligatory role for PKCδ in PIP 2‐mediated activation of store‐operated TRPC1 channels in vascular smooth muscle cells

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          Abstract

          Key points

          • In vascular smooth muscle cells (VSMCs), activation of Ca 2+‐permeable store‐operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca 2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease.

          • Activation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP 2). We investigated the identity of the PKC isoform involved in activating TRPC1‐based SOCs in rat mesenteric artery VSMCs.

          • TRPC1‐based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ‐dependent phosphorylation of TRPC1. Furthermore, generation of store‐operated interactions between PIP 2 and TRPC1 and activation of TRPC1‐based SOCs by PIP 2 required PKCδ.

          • These findings reveal that PKCδ activity has an obligatory role in activating TRPC1‐based SOCs, through regulating PIP 2‐mediated channel opening.

          Abstract

          In vascular smooth muscle cells (VMSCs), stimulation of Ca 2+‐permeable canonical transient receptor potential channel 1 (TRPC1)‐based store‐operated channels (SOCs) mediates Ca 2+ entry pathways that regulate cell contraction, proliferation and migration, which are processes associated with vascular disease. It is therefore important to understand how TRPC1‐based SOCs are activated. Stimulation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, with store‐operated PKC‐dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP 2). Experimental protocols used to activate TRPC1‐based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca 2+‐insensitive, which are characteristics of the novel group of PKC isoforms (δ, ε, η, θ). Hence, the present study examined whether a novel PKC isoform(s) is involved in activating TRPC1‐based SOCs in contractile rat mesenteric artery VSMCs. Store‐operated whole‐cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1‐based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store‐operated PIP 2 and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP 2‐mediated activation of TRPC1 currents. These results identify the involvement of PKCδ in stimulation of TRPC1‐based SOCs and highlight that store‐operated PKCδ activity is obligatory for channel opening by PIP 2, the probable activating ligand.

          Key points

          • In vascular smooth muscle cells (VSMCs), activation of Ca 2+‐permeable store‐operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca 2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease.

          • Activation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP 2). We investigated the identity of the PKC isoform involved in activating TRPC1‐based SOCs in rat mesenteric artery VSMCs.

          • TRPC1‐based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ‐dependent phosphorylation of TRPC1. Furthermore, generation of store‐operated interactions between PIP 2 and TRPC1 and activation of TRPC1‐based SOCs by PIP 2 required PKCδ.

          • These findings reveal that PKCδ activity has an obligatory role in activating TRPC1‐based SOCs, through regulating PIP 2‐mediated channel opening.

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          Store-Operated Calcium Channels.

          Store-operated calcium channels (SOCs) are a major pathway for calcium signaling in virtually all metozoan cells and serve a wide variety of functions ranging from gene expression, motility, and secretion to tissue and organ development and the immune response. SOCs are activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the first characterization of SOCs through electrophysiology, the identification of the STIM proteins as ER Ca(2+) sensors and the Orai proteins as store-operated channels has enabled rapid progress in understanding the unique mechanism of store-operate calcium entry (SOCE). Depletion of Ca(2+) from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca(2+) depletion is coupled to the activation of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation, Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE, and the functions of STIM and Orai in physiology and disease.
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            Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology.

            The Journal of Physiology and Experimental Physiology have always used UK legislation as the basis of their policy on ethical standards in experiments on non-human animals. However, for international journals with authors, editors and referees from outside the UK the policy can lack transparency and is sometimes cumbersome, requiring the intervention of a Senior Ethics Reviewer or advice from external experts familiar with UK legislation. The journals have therefore decided to set out detailed guidelines for how authors should report experimental procedures that involve animals. As well as helping authors, this new clarity will facilitate the review process and decision making where there are questions regarding animal ethics.
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              Transient receptor potential channels in the vasculature.

              The mammalian genome encodes 28 distinct members of the transient receptor potential (TRP) superfamily of cation channels, which exhibit varying degrees of selectivity for different ionic species. Multiple TRP channels are present in all cells and are involved in diverse aspects of cellular function, including sensory perception and signal transduction. Notably, TRP channels are involved in regulating vascular function and pathophysiology, the focus of this review. TRP channels in vascular smooth muscle cells participate in regulating contractility and proliferation, whereas endothelial TRP channel activity is an important contributor to endothelium-dependent vasodilation, vascular wall permeability, and angiogenesis. TRP channels are also present in perivascular sensory neurons and astrocytic endfeet proximal to cerebral arterioles, where they participate in the regulation of vascular tone. Almost all of these functions are mediated by changes in global intracellular Ca(2+) levels or subcellular Ca(2+) signaling events. In addition to directly mediating Ca(2+) entry, TRP channels influence intracellular Ca(2+) dynamics through membrane depolarization associated with the influx of cations or through receptor- or store-operated mechanisms. Dysregulation of TRP channels is associated with vascular-related pathologies, including hypertension, neointimal injury, ischemia-reperfusion injury, pulmonary edema, and neurogenic inflammation. In this review, we briefly consider general aspects of TRP channel biology and provide an in-depth discussion of the functions of TRP channels in vascular smooth muscle cells, endothelial cells, and perivascular cells under normal and pathophysiological conditions.
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                Author and article information

                Contributors
                aalbert@sgul.ac.uk
                Journal
                J Physiol
                J Physiol
                10.1111/(ISSN)1469-7793
                TJP
                jphysiol
                The Journal of Physiology
                John Wiley and Sons Inc. (Hoboken )
                0022-3751
                1469-7793
                21 July 2020
                15 September 2020
                21 July 2020
                : 598
                : 18 ( doiID: 10.1113/tjp.v598.18 )
                : 3911-3925
                Affiliations
                [ 1 ] Department of Pharmacology University of California 451, Health Sciences Drive, Suite 3503 Davis CA USA
                [ 2 ] Leeds Institute of Cardiovascular and Metabolic Medicine Faculty of Medicine and Health University of Leeds Leeds UK
                [ 3 ] Vascular Biology Research Centre Molecular and Clinical Research Institute St George's University of London Cranmer Terrace London UK
                Author notes
                [*] [* ] Corresponding author Anthony Albert: Vascular Biology Research Centre, Molecular and Clinical Research Institute, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK. Email: aalbert@ 123456sgul.ac.uk

                Author information
                https://orcid.org/0000-0001-9399-4013
                https://orcid.org/0000-0002-5850-8005
                https://orcid.org/0000-0002-3596-9634
                Article
                TJP14246
                10.1113/JP279947
                7656825
                32627185
                693f9dc4-8328-4e9c-bdca-0dd3cbf977bd
                © 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 29 April 2020
                : 01 July 2020
                Page count
                Figures: 11, Tables: 0, Pages: 15, Words: 9334
                Funding
                Funded by: Biotechnology and Biological Sciences Research Council , open-funder-registry 10.13039/501100000268;
                Award ID: BB/J007226/1
                Award ID: BB/M018350/1
                Categories
                Research Paper
                Molecular and Cellular
                Custom metadata
                2.0
                15 September 2020
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.9.3 mode:remove_FC converted:11.11.2020

                Human biology
                pkc,pip,store‐operated channels2,trpc1,vascular smooth muscle
                Human biology
                pkc, pip, store‐operated channels2, trpc1, vascular smooth muscle

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