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 fromTrpv4^−/−andAqp4^−/−mice exhibited suppressed transcription of genes encodingTrpv4,Aqp4, and theKir4.1subunit of inwardly rectifying potassium channels. Swelling and [Ca²⁺]_ielevations 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) orTrpv4ablation. Elimination ofAqp4suppressed swelling-induced [Ca²⁺]_ielevations but only modestly attenuated the amplitude of Ca²⁺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 expressingXenopusoocyte 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 Ca²⁺entry through TRPV4 channels reciprocally modulates volume regulation, swelling, andAqp4gene 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 STATEMENTWe 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 ofAqp4andKir4.1genes 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.

Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na⁺/K⁺/2Cl⁻cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.

Cerebrospinal fluid (CSF) envelops the brain and fills the central ventricles. This fluid is continuously replenished by net fluid extraction from the vasculature by the secretory action of the choroid plexus epithelium residing in each of the four ventricles. We have known about these processes for more than a century, and yet the molecular mechanisms supporting this fluid secretion remain unresolved. The choroid plexus epithelium secretes its fluid in the absence of a trans-epithelial osmotic gradient, and, in addition, has an inherent ability to secrete CSF against an osmotic gradient. This paradoxical feature is shared with other ‘leaky’ epithelia. The assumptions underlying the classical standing gradient hypothesis await experimental support and appear to not suffice as an explanation of CSF secretion. Here, we suggest that the elusive local hyperosmotic compartment resides within the membrane transport proteins themselves. In this manner, the battery of plasma membrane transporters expressed in choroid plexus are proposed to sustain the choroidal CSF secretion independently of the prevailing bulk osmotic gradient.