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David Krizaj, Daniel Ryskamp, Andrew Jo, Alan Verkman, Nanna Macaulay; Molecular coupling between TRPV4 and aquaporin 4 channels mediates osmosensation in Müller glia. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2673.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the molecular mechanism of volume regulation in Müller glia, radial astroglia that are central to the control of activity-dependent water fluxes in the vertebrate retina. A combination of biophysical, genetic and physiological approaches was used to characterize reciprocal interactions between the osmosensor TRPV4 and the water channel aquaporin 4 (AQP4) in heterologously expressing Xenopus oocytes, and in Müller glia from wild type and AQP4 knockout mice.
Cell area, volume and calcium concentration [Ca2+]i were measured under isotonic and hypotonic conditions from wild type and AQP4-/- Müller glia loaded with fluorescent markers of cell volume and intracellular calcium. Transmembrane currents in Xenopus oocytes transfected with combinations of TRPV4, AQP1 and/or AQP4 channels were measured with the two-electrode voltage clamp under isotonic conditions, during mild hypotonic stimulation (HTS) and/or stimulation with selective modulators of TRPV4 function. Stretch-induced currents from outside-out membrane patches were recorded with high-speed pressure clamp.
TRPV4 and AQP4 colocalized in Müller glial endfeet and radial processes. HTS- evoked cation influx and volume increase in Müller cells were sensitive to BAPTA and TRPV4 blockers whereas HTS-induced changes in volume regulation, kinetics of hypotonically induced [Ca2+]i signals, calcium signal amplitudes and the dose-[Ca2+]i response relationship were compromised in mice lacking AQP4. Oocytes co-transfected with TRPV4 and AQP1/AQP4 exhibited a similar rate of swelling (13.0 + 1.5%/100 sec vs. 14.1+1.3%/100 sec), however, HTS facilitated cation fluxes in TRPV4-expressing cells that co-expressed AQP4, but not AQP1. This effect was sensitive to TRPV4 antagonists, indicating that it was mediated through TRPV4 channels. Consistent with this, TRPV4 agonist-induced Ca entry into transfected oocytes was augmented by the presence of AQP4.
Our data suggest that a macromolecular complex consisting of a stretch sensor and a water channel (TRPV4 and AQP4) regulates osmotically-induced cation fluxes in Müller glia. The complex senses and responds to changes in osmolarity, rather than changes in cell volume. TRPV4-AQP4 channels are strategically localized to Müller endfeet where they may regulate retina:blood fluxes driven by the metabolism, intraocular pressure and retinal pathologies.
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