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Anthony Iuso, Andrew Jo, Oleg Yarishkin, Alen Delic, David Krizaj; Membrane Cholesterol Differentially Regulates TRPV4 Drug-Channel Efficacy and Osmotic-Evoked Swelling in Müller Astroglia. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6425.
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© ARVO (1962-2015); The Authors (2016-present)
The polymodal TRPV4 cation channel is a promiscuous cellular sensor capable of integrating various stimuli, such as temperature, osmolality and tensile forces from the extracellular milieu. However, how these diverse stimuli regulate channel gating and what role, if any, the lipid bilayer has in the regulation of gating, has been a long-standing question in the field. To address it, we studied how TRPV4 activation in retinal neurons, Müller glia and heterologous HEK293 overexpressors – induced through pharmacological agents, hypotonicity and heat – is impacted following the sequestration of membrane cholesterol with methyl-β-cyclodextrin (mβCD).
Membranous cholesterol was removed from TRPV4-OE HEK293 cells and dissociated retinal cells via incubation with 10mM mβCD or filipin (4 ug/ml). In a subset of experiments, the cholesterol content was replenished by incubating cells with cholesterol-loaded-mβCD (1:10mM). Optical Ca2+ imaging with ratiometric indicator Fura-2 (10μM) was used to measure [Ca2+]i. The extent of cell swelling due to osmotic challenge was determined by changes in fluorescence resulting from intracellular volume changes.
Lowering membrane sterol content was sufficient to attenuate agonist (GSK1016790A) and hypotonicity-evoked [Ca2+]i elevations in TRPV4-OE HEK293 cells and Müller cells, whereas responses to heat were unaffected. Ca2+ signaling deficits were rescued by cholesterol restoration delivered through cholesterol:mβCD complex substitution, arguing against nonspecific consequences of depletion. In the absence of membrane cholesterol, TRPV4 antagonist (HC067047) lost its effectiveness as an inhibitor of hypotonically evoked, TRPV4-mediated [Ca2+]i responses, which however were blocked by the nonspecific Ca2+ channel pore blocker, gadolinium. Cholesterol-depleted Müller cells exhibited a reduced capacity for swelling, slow swelling kinetics and an absence of regulatory volume decrease.
Our findings suggest that TRPV4 activation requires the formation of local, cholesterol-enriched, lipid microdomains that are likely to regulate the energy barrier for conformational switches effected by pharmacological agents and hypo-osmotic stretch. We conclude that cholesterol saturation and the membrane lipid environment are important regulators of retinal-glial TRPV4 signaling, calcium homeostasis and volume regulation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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