AQP4-independent TRPV4 modulation of plasma membrane water permeability

Abstract

Despite of the major role of aquaporin (AQP) water channels in controlling transmembrane water fluxes, alternative ways for modulating water permeation have been proposed. In the Central Nervous System (CNS), Aquaporin-4 (AQP4) is reported to be functionally coupled with the calcium-channel Transient-Receptor Potential Vanilloid member-4 (TRPV4), which is controversially involved in cell volume regulation mechanisms and water transport dynamics. The present work aims to investigate the selective role of TRPV4 in regulating plasma membrane water permeability in an AQP4-independent way. Fluorescence-quenching water transport experiments in Aqp4–/– astrocytes revealed that cell swelling rate is significantly increased upon TRPV4 activation and in the absence of AQP4. The biophysical properties of TRPV4-dependent water transport were therefore assessed using the HEK-293 cell model. Calcein quenching experiments showed that chemical and thermal activation of TRPV4 overexpressed in HEK-293 cells leads to faster swelling kinetics. Stopped-flow light scattering water transport assay was used to measure the osmotic permeability coefficient (Pf, cm/s) and activation energy (Ea, kcal/mol) conferred by TRPV4. Results provided evidence that although the Pf measured upon TRPV4 activation is lower than the one obtained in AQP4-overexpressing cells (Pf of AQP4 = 0.01667 ± 0.0007; Pf of TRPV4 = 0.002261 ± 0.0004; Pf of TRPV4 + 4αPDD = 0.007985 ± 0.0006; Pf of WT = 0.002249 ± 0.0002), along with activation energy values (Ea of AQP4 = 0.86 ± 0.0006; Ea of TRPV4 + 4αPDD = 2.73 ± 1.9; Ea of WT = 8.532 ± 0.4), these parameters were compatible with a facilitated pathway for water movement rather than simple diffusion. The possibility to tune plasma membrane water permeability more finely through TRPV4 might represent a protective mechanism in cells constantly facing severe osmotic challenges to avoid the potential deleterious effects of the rapid cell swelling occurring via AQP channels.

The deletion of AQP4 and TRPV4 affects astrocyte swelling/volume recovery in response to ischemia-mimicking pathologies

“…Our results showed that deletion of AQP4 does not affect astrocyte swelling under hypoosmotic stress. This result is not surprising, because it agrees with the results of Murphy et al (2017) , Barile et al (2023) , who reported that the swelling of astrocytes does not require AQP4. Similarly, Mola et al (2016) showed that cells lacking AQP4 can swell, albeit more slowly, but are unable to trigger the RVD.…”

Osmosensing in trabecular meshwork cells

“…4 & 5) and pressure-evoked current (Yarishkin, Phuong et al 2021) are consistent with time-dependent formation of EET messenger molecules (Vriens, Watanabe et al 2004, Ryskamp, Frye et al 2016). TRPV4-mediated osmosensing has been associated with the promotion of cell swelling and RVD (Becker, Blase et al 2005, Hoffmann, Lambert et al 2009, Jo, Ryskamp et al 2015, Barile, Mola et al 2023. The suppression of the amplitude of HTS-induced swelling by HC-06 and stimulation of cell volume expansion by GSK101 under isotonic conditions suggest that, as reported recently for glial cells (Jo, Ryskamp et al 2015, Mola, Sparaneo et al 2016, Barile, Mola et al 2023 TRPV4 activity promotes cell swelling instead of time-dependent volume regulation.…”

“…Given that (i) TRPV4 inhibition, knockdown and Ca 2+ chelation suppress HTS-induced swelling (Fig. 2) (Jo, Ryskamp et al 2015) and (ii) swelling is augmented in cells that overexpress TRPV4 (Barile, Mola et al 2023), AQPs may not be required to facilitate cell swelling in response TRPV4-mediated Ca 2+ influx. NKCC1 transports water (Zeuthen and MacAulay 2002) and may modulate outflow resistance (Al-Aswad, Gong et al 1999) whereas TRPM4 shows strong expression in mouse and human TM and mediates 9-phenanthrol-sensitive Na + influx in GSK101-stimulated pTM cells (Yarishkin, Phuong et al 2022).…”

“…TRPV4 modulates systemic osmoregulation (Liedtke and Friedman 2003), water diffusion and edema formation in the retina, lung and brain (Hoshi, Okabe et al 2018, Orduna Rios, Noguez Imm et al 2019, Sonkusare and Laubach 2022 but its role in volume regulation has been under debate, with studies implicating it in the promotion of swelling (Jo, Ryskamp et al 2015, Barile, Mola et al 2023) and regulatory volume decrease (RVD) (Becker, Blase et al 2005, Hoffmann, Lambert et al 2009, Jo, Ryskamp et al 2015. Here, we identify TRPV4 as a central transducer of swelling-induced calcium signaling in human TM cells, show that its activation contributes to volume expansion, and elucidate its collaboration with Clefflux as a component of the swelling-evoked current ISwell.…”

Aquaporin-4 and Parkinson’s Disease

“…AQP4 is known to be functionally and structurally associated with the volume-and thermosensitive calcium channel transient receptor potential vanilloid member-4 (TRPV4), which is also implicated in mechanisms regulating water transport and calcium homeostasis, the detection of changes in cell volume, and possibly, its regulation [144][145][146][147]. In vitro studies relating to Aqp4 -/astrocytes revealed that the possible role of TRPV4 is to tune plasma membrane water permeability and protect against severe osmotic challenges due to fast AQP4-dependent water transport [146]. However, the role of TRPV4 and calcium influx from extracellular space in the regulation of cell volume is controversial, and there is evidence that TRPV4 is not essential for regulatory volume decreases after AQP4-mediated cell swelling [145,147].…”