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TRPV4 and AQP4 Channels Synergistically Regulate Cell Volume and Calcium Homeostasis in Retinal Müller Glia.
Jo AO
,
Ryskamp DA
,
Phuong TT
,
Verkman AS
,
Yarishkin O
,
MacAulay N
,
Križaj D
.
???displayArticle.abstract??? 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 from Trpv4(-/-) and Aqp4(-/-) mice exhibited suppressed transcription of genes encoding Trpv4, Aqp4, and the Kir4.1 subunit of inwardly rectifying potassium channels. Swelling and [Ca(2+)]i elevations 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) or Trpv4 ablation. Elimination of Aqp4 suppressed swelling-induced [Ca(2+)]i elevations but only modestly attenuated the amplitude of Ca(2+) 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 expressing Xenopus oocyte 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 Ca(2+) entry through TRPV4 channels reciprocally modulates volume regulation, swelling, and Aqp4 gene 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 statement: We 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 of Aqp4 and Kir4.1 genes 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.
Figure 1. TRPV4 and AQP4 colocalize in the mouse inner retina, yet TRPV4 and AQP4 trafficking are independent of each other. Ai, Vertical cryosections of WT mouse retinas immunolabeled for TRPV4 (Alexa Fluor 488) and AQP4 (Alexa Fluor 594) show preferential colocalization in Müller end feet and proximal processes entering the IPL (arrows). RGC somata are TRPV4 immunopositive and do not express AQP4 (arrowheads). Aii, Aiii, Higher magnification of Müller end feet double labeled for Kir4.1 and GS, and TRPV4 and Kir4.1, respectively. Nuclei labeled with DAPI. GCL, Ganglion cell layer; INL, inner nuclear layer. Scale bars, 20 μm. BiâBiii, Trpv4â/â retina. GS and Kir4.1 signals in KO tissue are indistinguishable from WT controls, whereas AQP4 signals appear to be less pronounced. Biv, Limited nonspecific TRPV4 immunoreactivity in Trpv4â/â retina costained for Kir4.1. C, Aqp4â/â retinas. Ci, TRPV4 and GS colocalize in glial end feet at the bloodâretina barrier, whereas RGC somata are immunoreactive for TRPV4. Cii, Double labeling for GS and Kir4.1 shows normal targeting to glial processes and end feet. Ciii, GFAP is expressed in astrocyte processes but is absent from GS immunoreactivity in Müller processes and end feet, indicating absence of reactive gliosis. Scale bars, 20 μm.
Figure 2. Expression of Trpv4, Aqp4, and Kir4.1 transcripts is effected by genetic elimination of TRPV4 or AQP4. Semiquantitative RT-PCR analysis of mRNA levels in WT, Trpv4â/â, and Aqp4â/â mouse retinas. Fold change in mRNA expression was calculated relative to basal after normalization of expression to α-tubulin (Tuba1a) mRNA. The relative abundance of Trpv4 transcripts is decreased in Aqp4â/â samples and vice versa. Kir4.1 transcript levels are lower in both Trpv4â/â and Aqp4â/â retinas, whereas the abundance of presumed housekeeping genes Glul, Actb, Gapdh, and 18S rRNA (the 18S structural subunit of the small eukaryotic 40S ribosomal subunit, 18S-rRNA) mRNAs was unaffected by the deletion of Trpv4 or Aqp4.
Figure 3. Figure 6.
AQP4 regulates the amplitude and kinetics of the swelling-induced TRPV4-mediated transmembrane current. A, B, Representative time course of the HTS (235 mOsm)-evoked whole-cell current in WT (black traces) and Aqp4â/â (blue traces) Müller cells at positive (100 mV; open circles) and negative (â100 mV; filled circles) holding potentials. C, Averaged I-V relationship of HTS-induced currents in WT (white; n = 14) and Aqp4â/â (blue; n = 13) Müller cells, WT cells treated with HC-06 (gray; n = 5), and Aqp4â/â cells treated with HC-06 (red; n = 8). Individual IâV curves of HTS-induced currents were subtracted from pre-HTS baselines. HC-06 was applied 4 min before the application of HTS and was present throughout the experiment. D, Summary for HTS-induced currents at 100 mV (open bars) and â100 mV (shadowed bars) for WT (white) and Aqp4â/â (blue) Müller cells, WT cells treated with HC-06 (gray), and Aqp4â/â cells treated with HC-06.
Figure 4.
TRPV4-induced Ca2+ entry regulates Müller glial swelling and RVD. A, Cell volume changes in a fura-5F-loaded Müller cell. The sum of 340/380 emissions, adjusted to yield calcium-insensitive intensity values, shows dose-dependent decreases as cell volume increases in the presence of HTS. RVD is observed during the 140 mOsm (46.67% tonicity) but not 190 mOsm (63.33% tonicity) stimulus (arrowhead). B, Doseâresponse curves for changes in peak perikaryal cross-sectional area in the presence of osmotic challenge show reduced glial swelling in Trpv4â/â (n = 25â31 cells per stimulus) but not substantially in Aqp4â/â cells (n = 31â71) compared with WT cells (n = 27â61). Removal of extracellular Ca2+ during swelling of WT cells (13.3% tonicity) shifted swelling to the extrapolated Trpv4â/â curve (light gray data point; n = 27). Inset, Volume measurement (340 + 380 signals, normalized to baseline) in WT cells (n = 8) exposed to extreme HTS (40 mOsm) and shown with inverted traces. Note the lower fluorescence decrease in Ca2+-free saline and the absence of RVD under these conditions (arrow). C, The time course of swelling in WT, Trpv4â/â, and Aqp4â/â cells during a 5 min exposure to 140 mOsm HTS, normalized to base and peak cross-sectional areas to compare swelling kinetics. D, RVD during exposure to 140 mOsm HTS is impaired in Trpv4â/â (n = 38) and Aqp4â/â (n = 57) cells compared with WT cells (n = 52). E, WT and Trpv4â/â cell area over time normalized to baseline (20% peak swelling; 190 mOsm for WT, n = 9; 140 mOsm for Trpv4â/â cells, n = 8). F, WT, Trpv4â/â, and Aqp4â/â cell area over time normalized to baseline (40% peak swelling; 140 mOsm for WT, n = 10; 90 mOsm for Trpv4â/â cells, n = 10; 140 mOsm for Aqp4â/â cells, n = 5).
Figure 5.
TRPV4 blockers suppress the swelling of Aqp4−/− Müller glia loaded with calcein AM. A, The TRPV4 antagonist HC-06 (1 μm) suppresses HTS-induced increases in Aqp4−/− glial volume (190 mOsm; n = 9 cells from 5 slides). B, Cumulative data for cell volume measurements in Aqp4−/− cells stimulated with 190 mOsm in the presence/absence of HC-06 (n = 13 and 20, respectively). C, Cell area measurements revealed a similar extent of swelling after 5 min of HTS (190 mOsm) perfusion in WT (n = 24) and Aqp4−/− (n = 23) Müller cells. HC-06 (1 μm) significantly decreased the extent of HTS-induced glial swelling in WT (n = 10) and Aqp4−/− (n = 12) Müller cells.
Figure 6.
AQP4 regulates the amplitude and kinetics of the swelling-induced TRPV4-mediated transmembrane current. A, B, Representative time course of the HTS (235 mOsm)-evoked whole-cell current in WT (black traces) and Aqp4â/â (blue traces) Müller cells at positive (100 mV; open circles) and negative (â100 mV; filled circles) holding potentials. C, Averaged I-V relationship of HTS-induced currents in WT (white; n = 14) and Aqp4â/â (blue; n = 13) Müller cells, WT cells treated with HC-06 (gray; n = 5), and Aqp4â/â cells treated with HC-06 (red; n = 8). Individual IâV curves of HTS-induced currents were subtracted from pre-HTS baselines. HC-06 was applied 4 min before the application of HTS and was present throughout the experiment. D, Summary for HTS-induced currents at 100 mV (open bars) and â100 mV (shadowed bars) for WT (white) and Aqp4â/â (blue) Müller cells, WT cells treated with HC-06 (gray), and Aqp4â/â cells treated with HC-06.
Figure 7.
AQP4 amplifies glial HTS-induced TRPV4 activation with moderate (140 mOsm) but not extreme (90 mOsm) HTS. A, B, Ca2+ signals in response to 140 mOsm HTS were blocked in Trpv4â/â (n = 50) and Aqp4â/â (n = 64) Müller cells compared with WT cells (n = 20). HC-06 inhibited HTS-induced [Ca2+]i elevations in WT cells (n = 14) but had no additional effect in Aqp4â/â cells (n = 7), indicating that TRPV4 activation by HTS requires Aqp4. C, At excessively large hypotonic gradients (90 mOsm), Aqp4â/â cells (blue bar) displayed robust HTS-induced [Ca2+]i responses approaching those of the WT cells. Trpv4â/â cells (red bar) showed no [Ca2+]i response when challenged with HTS.
Figure 8.
AQP4 channels amplify stretch-induced TRPV4 activation. A, Oocytes expressing TRPV4 and AQP4 alone or in combination were voltage clamped at Vm = â30 mV and exposed to 100 nm GSK101 (indicated by black bar). After 60 s GSK exposure, an IâV curve was obtained by jumping the clamp potential to potentials ranging from 60 to â140 mV at 20 mV intervals (seen as the vertical lines in the current trace). B, Representative IâV relations from oocytes expressing TRPV4 + AQP4, TRPV4, or AQP4 in control solution (left), in GSK101 solutions (right), or when exposed to Î125 mOsm hypotonic challenge (middle). C, Summarized I-V relations obtained with GSK101 (as in B; n = 5â8). D, Representative current (black lines) and volume (gray lines) traces recorded simultaneously from oocytes expressing TRPV4 and AQP4 alone or in combination during exposure to a Î125 mOsm hypotonic stimulus (indicated by the black bar; n = 5â9). E, Summarized I-V relations obtained during Î125 mOsm. F, Oocytes expressing TRPV4 alone or in combination with AQP4 were exposed to hypotonic challenges of different magnitudes to observe similar degree of cell swelling (Î125 mOsm for TRPV4-expressing oocytes and Î15 mOsm for TRPV4/AQP4-expressing oocytes), and IâV relations were obtained after 60 s of cell swelling (n = 8â9).
Figure 9.
Proposed schema of TRPV4–AQP4 interactions in the Müller cell end foot. Hypotonic stress stimulates fluxes of water after the osmotic gradient. The resulting increase in cell volume stretches the plasma membrane, activating TRPV4 and a Ca2+- and stretch-sensitive PLA2. The product, arachidonic acid (AA), is a precursor for the cytochrome P450-mediated synthesis of eicosanoid metabolites (EETs) that serve as final activators of TRPV4 channels (Ryskamp et al., 2015) but may also suppress Kir4.1 (Bringmann et al., 1998). The expression of Kir4.1 (Reichenbach and Bringmann, 2010) and reactive gliosis (Ryskamp et al., 2014) are modulated by HTS-induced influx of Ca2+. It remains to be determined whether stretch activates PLA2 simultaneously with TRPV4 or whether its activation, which is Ca2+ dependent, amplifies the initial TRPV4 signal (blue arrows). Ca2+ induces both cell swelling (short term) and RVD (long term) and may be required for Ca2+-dependent gene expression (Aqp4, Kir4.1, Trpv4, Gfap). High levels of Ca2+ may also stimulate the activity of BK and VRAC channels and facilitate RVD.
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