title: TRPV4 and AQP4 Channels Synergistically Regulate Cell Volume and Calcium Homeostasis in Retinal Müller Glia.
abstract: Brain edema formation occurs after dysfunctional control of extracellular volume partly through impaired astrocytic ion and water transport. Here, we show that such processes might involve synergistic cooperation between the glial water channel aquaporin 4 (AQP4) and the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel. In mouse retinas, TRPV4 colocalized with AQP4 in the end feet and radial processes of Müller astroglia. Genetic ablation of TRPV4 did not affect the distribution of AQP4 and vice versa. However, retinas from Trpv4(-/-) and Aqp4(-/-) mice exhibited suppressed transcription of genes encoding Trpv4, Aqp4, and the Kir4.1 subunit of inwardly rectifying potassium channels. Swelling and [Ca(2+)]i elevations evoked in Müller cells by hypotonic stimulation were antagonized by the selective TRPV4 antagonist HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) or Trpv4 ablation. Elimination of Aqp4 suppressed swelling-induced [Ca(2+)]i elevations but only modestly attenuated the amplitude of Ca(2+) signals evoked by the TRPV4 agonist GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide]. Glial cells lacking TRPV4 but not AQP4 showed deficits in hypotonic swelling and regulatory volume decrease. Functional synergy between TRPV4 and AQP4 during cell swelling was confirmed in the heterologously expressing Xenopus oocyte model. Importantly, when the swelling rate was osmotically matched for AQP4-positive and AQP4-negative oocytes, TRPV4 activation became independent of AQP4. We conclude that AQP4-mediated water fluxes promote the activation of the swelling sensor, whereas Ca(2+) entry through TRPV4 channels reciprocally modulates volume regulation, swelling, and Aqp4 gene expression. Therefore, TRPV4-AQP4 interactions constitute a molecular system that fine-tunes astroglial volume regulation by integrating osmosensing, calcium signaling, and water transport and, when overactivated, triggers pathological swelling. Significance statement: We characterize the physiological features of interactions between the astroglial swelling sensor transient receptor potential isoform 4 (TRPV4) and the aquaporin 4 (AQP4) water channel in retinal Müller cells. Our data reveal an elegant and complex set of mechanisms involving reciprocal interactions at the level of glial gene expression, calcium homeostasis, swelling, and volume regulation. Specifically, water influx through AQP4 drives calcium influx via TRPV4 in the glial end foot, which regulates expression of Aqp4 and Kir4.1 genes and facilitates the time course and amplitude of hypotonicity-induced swelling and regulatory volume decrease. We confirm the crucial facets of the signaling mechanism in heterologously expressing oocytes. These results identify the molecular mechanism that contributes to dynamic regulation of glial volume but also provide new insights into the pathophysiology of glial reactivity and edema formation.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Moran Eye Institute.
authors.forename: Andrew O
authors.initials: AO
authors.lastname: Jo
authors.affiliation: Department of Ophthalmology and Visual Sciences, Moran Eye Institute, Interdepartmental Program in Neuroscience, and david.krizaj@hsc.utah.edu daniel.ryskamp@utsouthwestern.edu.
authors.forename: Daniel A
authors.identifier: http://orcid.org/0000-0002-2197-0730
authors.initials: DA
authors.lastname: Ryskamp
authors.affiliation: Department of Ophthalmology and Visual Sciences, Moran Eye Institute.
authors.forename: Tam T T
authors.initials: TT
authors.lastname: Phuong
authors.affiliation: Department of Medicine, University of California San Francisco, San Francisco, California 94143, and.
authors.forename: Alan S
authors.initials: AS
authors.lastname: Verkman
authors.affiliation: Department of Ophthalmology and Visual Sciences, Moran Eye Institute.
authors.forename: Oleg
authors.identifier: http://orcid.org/0000-0002-8215-7444
authors.initials: O
authors.lastname: Yarishkin
authors.affiliation: Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Nanna
authors.initials: N
authors.lastname: MacAulay
authors.affiliation: Department of Ophthalmology and Visual Sciences, Moran Eye Institute, Interdepartmental Program in Neuroscience, and Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 84132, david.krizaj@hsc.utah.edu daniel.ryskamp@utsouthwestern.edu.
authors.forename: David
authors.identifier: http://orcid.org/0000-0003-4468-3029
authors.initials: D
authors.lastname: Križaj
mesh_terms: D000818:Animals; D051401:Aquaporin 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D048429:Cell Size; D015870:Gene Expression / Q000235:genetics / Q000502:physiology; D006706:Homeostasis / Q000502:physiology*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009025:Morpholines / Q000494:pharmacology; D009457:Neuroglia / Q000502:physiology* / Q000648:ultrastructure; D009865:Oocytes / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000502:physiology; D011758:Pyrroles / Q000494:pharmacology; D012160:Retina / Q000166:cytology / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D014867:Water / Q000378:metabolism; D014981:Xenopus; D000098854:Kcnj10 Channel
chemical_list: C496031:Aqp4 protein, mouse; D051401:Aquaporin 4; C573308:HC-067047; D000098854:Kcnj10 Channel; D009025:Morpholines; C530602:N-(1-((4-(2-(((2,4-dichlorophenyl)sulfonyl)amino)-3-hydroxypropanoyl)-1-piperazinyl)carbonyl)-3-methylbutyl)-1-benzothiophene-2-carboxamide; D024661:Potassium Channels, Inwardly Rectifying; D011758:Pyrroles; D013449:Sulfonamides; D050916:TRPV Cation Channels; C486576:Trpv4 protein, mouse; D014867:Water; D007930:Leucine; D002118:Calcium
keywords: Muller cell; TRPV4; aquaporin 4; osmoregulation; retina; swelling