Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K+ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (Vm), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in Vm. Cell volume and Vm changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by Vm depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K+ or Cl− channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in Vm– define the success of RVD.
Neural activity alters osmotic gradients favoring cell swelling in retinal Müller cells. This swelling is followed by a regulatory volume decrease (RVD), partially mediated by an efflux of KCl and water. The transient receptor potential channel 4 (TRPV4), a nonselective calcium channel, has been proposed as a candidate for mediating intracellular Ca elevation induced by swelling. We previously demonstrated in a human Müller cell line (MIO-M1) that RVD strongly depends on ion channel activation and, consequently, on membrane potential (V ). The aim of this study was to investigate if Ca influx via TRPV4 contributes to RVD by modifying intracellular Ca concentration and/or modulating V in MIO-M1 cells. Cell volume, intracellular Ca levels, and V changes were evaluated using fluorescent probes. Results showed that MIO-M1 cells express functional TRPV4 which determines the resting V associated with K channels. Swelling-induced increases in Ca levels was due to both Ca release from intracellular stores and Ca influx by a pathway alternative to TRPV4. TRPV4 blockage affected swelling-induced biphasic response (depolarization-repolarization), suggesting its participation in modulating V changes during RVD. Agonist stimulation of Ca influx via TRPV4 activated K channels hyperpolarizing V and accelerating RVD. We propose that TRPV4 forms a signaling complex with Ca and/or voltage-dependent K channels to define resting V and V changes during RVD. TRPV4 involvement in RVD depends on the type of stimuli and/or degree of channel activation, leading to a maximum RVD response when Ca influx overcomes a threshold and activates further signaling pathways in cell volume regulation. J. Cell. Biochem. 118: 2302-2313, 2017. © 2017 Wiley Periodicals, Inc.
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