title: Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels.
abstract: The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca(2+) signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca(2+)-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca(2+) release-activated currents (ICRAC) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca(2+) waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER-mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia.
  
  
  CONCLUSIONS
  Store-operated Ca(2+) signaling represents a major signaling pathway and source of cytosolic Ca(2+) in astrocytes. Here, we show that the store-operated response in Müller cells, radial glia that perform key structural, signaling, osmoregulatory, and mechanosensory functions within the retina, is mediated through synergistic activation of transient receptor potential and Orai channels. The end-foot disproportionately expresses the depletion sensor stromal interacting molecule 1, which contains an extraordinarily high density of endoplasmic reticulum cisternae that shadow neuronal, astrocytic, vascular, and axonal structures; interface with mitochondria; but also originate store-operated Ca(2+) entry-induced transcellular Ca(2+) waves that propagate glial excitation into the proximal retina. These results identify a molecular mechanism that underlies complex interactions between the plasma membrane and calcium stores, and contributes to astroglial function, regulation, and response to mechanical stress.
authors.affiliation: Departments of Ophthalmology & Visual Sciences.
authors.forename: Tünde
authors.initials: T
authors.lastname: Molnár
authors.affiliation: Departments of Ophthalmology & Visual Sciences.
authors.forename: Oleg
authors.identifier: http://orcid.org/0000-0002-8215-7444
authors.initials: O
authors.lastname: Yarishkin
authors.affiliation: Departments of Ophthalmology & Visual Sciences.
authors.forename: Anthony
authors.initials: A
authors.lastname: Iuso
authors.affiliation: Departments of Ophthalmology & Visual Sciences.
authors.forename: Peter
authors.identifier: http://orcid.org/0000-0002-8166-3476
authors.initials: P
authors.lastname: Barabas
authors.affiliation: Departments of Ophthalmology & Visual Sciences.
authors.forename: Bryan
authors.identifier: http://orcid.org/0000-0001-5527-6643
authors.initials: B
authors.lastname: Jones
authors.affiliation: Departments of Ophthalmology & Visual Sciences.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Marc
authors.affiliation: Departments of Ophthalmology & Visual Sciences.
authors.forename: Tam T T
authors.initials: TT
authors.lastname: Phuong
authors.affiliation: Departments of Ophthalmology & Visual Sciences, Neurobiology & Anatomy, and Bioengineering, University of Utah School of Medicine, Salt Lake City, Utah 84132 david.krizaj@hsc.utah.edu.
authors.forename: David
authors.identifier: http://orcid.org/0000-0003-4468-3029
authors.initials: D
authors.lastname: Križaj
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D004721:Endoplasmic Reticulum / Q000378:metabolism; D063928:Ependymoglial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009798:Ocular Hypertension / Q000139:chemically induced / Q000473:pathology; D011720:Pyrazoles / Q000494:pharmacology; D012160:Retina / Q000166:cytology; D000071737:Stromal Interaction Molecule 1; D000071738:Stromal Interaction Molecule 2; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: C583716:2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide; D001549:Benzamides; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005904:Glial Fibrillary Acidic Protein; D008562:Membrane Glycoproteins; D011720:Pyrazoles; C100757:Stim1 protein, mouse; C525024:Stim2 protein, mouse; D000071737:Stromal Interaction Molecule 1; D000071738:Stromal Interaction Molecule 2; D050052:TRPC Cation Channels; C491858:Trpc2 protein, mouse; C096351:transient receptor potential cation channel, subfamily C, member 1; D002118:Calcium
keywords: ER stores; TRPC; astrocyte end-feet; calcium; reactive gliosis; store-operated channels