Toft-Bertelsen TL , Yarishkin O , Redmon S , Phuong TTT , Križaj D , MacAulay N .

P30 EY014800 NEI NIH HHS , R01 EY022076 NEI NIH HHS , R01 EY027920 NEI NIH HHS

             osmosensor activity

	Figure 1 – TRPV4 is activated by increased cell volume independently of PLA2 activity. (A) Representative confocal laser scanning micrographs of an uninjected oocyte (top) and an oocyte expressing TRPV4+AQP4 (bottom) after immunolabeling with phalloidin, anti-AQP4 and anti-TRPV4 antibodies confirmed the plasma membrane expression. (Ai) Representative volume- and current traces obtained from oocytes voltage clamped at Vm = -20 mV and challenged with a hyposmotic gradient (-100mOsm, indicated by a blue bar). Current traces were recorded with a 200 ms step protocol from an uninjected oocyte (far left) and oocytes expressing either AQP4 (middle left), TRPV4 (middle right) or TRPV4+AQP4 (far right). (B-H) Summarized I/V curves from TRPV4- and TRPV4+AQP4-expressing oocytes in control solution (black) or during application of a hyposmotic solution (red) without drug, or in control solution and hyposmotic solution with sc-3034 (PLA2 activator) (B,C; control solution in white, hyposmotic solution in blue), externally added PUFAs (D; control solution in white, hyposmotic solution in light purple), microinjected PUFAs (E,F; control solution in white, hyposmotic solution in light purple), ONO-RS-082 (PLA2 inhibitor) (G; control solution in white, hyposmotic solution in dark purple), or pBPB (PLA2 inhibitor) (H; control solution in white, hyposmotic solution in dark purple). Inserts: TRPV4-mediated current activity at -85 mV obtained after exposure to -100mOsm (red), in control solution with drug (white) and after exposure to -100 mOsm with drug (blue, light or dark purple) was normalized to that obtained in control condition without drug. N.S. = p > 0.05; one-way ANOVA, n = 9-10 oocytes.
	Figure 2 – No changes in swelling-induced activation of TRPV4 upon phosphorylation. (A-F) Representative I/V curves of TRPV4-mediated activity in control solution or in hyposmotic solution. Control solution is shown in black and hyposmotic (-100 mOsm) in red. (A-C) represents activity with PKA-, PKC-, or PKG activators (right panel). (D-F) represents activity with PKA-, PKC-, or PKG inhibitors (right panel). (G) Current activity, I, at -85 mV obtained after exposure to -100mOsm is shown as bars. Filled red bars correspond to HTS-induced TRPV4-mediated activity without drug, and open bars to the response with drug. (H) Experimental paradigm: Application of a hyposmotic gradient is indicated by a blue bar, and drug application by a pink bar. Arrows indicate when current activity was recorded. N.S. = p > 0.05, one-way ANOVA, n = 9-12 oocytes.
	Figure 3 – Cytoskeletal rearrangements are not required for activation of TRPV4. (A) Experimental paradigm and representative volume traces (top): Application of a hyposmotic gradient is indicated by a blue bar, and drug application by a pink bar. Representative current traces (bottom) recorded as indicated by arrows (in control and hyposmotic solutions before drug application, after recovery and after Lat. A and Taxol application). (B) Summarized I/V curves with control solution (black), hyposmotic solution (red), in control solution (white) and hyposmotic solution (light purple) after Lat.A/Taxol application. Inserts: TRPV4-mediated current activity at -85 mV obtained after exposure to -100mOsm (red), in control solution with Lat.A/Taxol (white) or in hyposmotic solution with Lat.A/Taxol (light purple) was normalized to that obtained in control condition. N.S. = p > 0.05; one-way ANOVA, n = 12 oocytes.
	Figure 4 – Related TRPVs are differentially gated. (A-C) Schematics of the phylogenetically related TRPV4 (top), TRPV1 (middle) and the V1 splice variant, VR.5’sv (bottom). (Ai –Ci) Summarized I/V curves from TRPV4- (top), TRPV1- (middle) or VR.5’sv-expressing oocytes (bottom) with control solution (black) and hyposmotic solution (red), or hyperosmotic solution (orange) or capsaicin (grey) (Aii-Cii). Inserts: Summarized I/V curves from TRPV4-, AQP4- (top), TRPV1- (middle) and VR.5’sv-expressing oocytes (bottom). Current activity at -85 mV obtained after exposure to -100mOsm, +100 mOsm and capsaicin were normalized to that obtained in control condition. N.S. = p > 0.05, * = p< 0.05, ** = p< 0.01 *** = p< 0.001; Students paired t-test (Ai-Ci, Aii), one-way ANOVA (Bii-Cii), n = 9 oocytes.
	Figure 5 – The N-terminus of TRPV4 is essential for the directionality of the volume-sensing. (A,B) Schematics of the constructed chimeras (top) with the N-terminus from TRPV1 (left) and the TRPV1 splice variant VR.5’sv (right), and micrographs of the chimeras immunolabeled with phalloidin, anti-AQP4 and anti-TRPV4 antibodies confirming plasma membrane expression. (C, D) Summarized I/V curves from TRPV4:TRPV1- (C) or TRPV4:VR.5’sv-expressing oocytes (D) with control solution (black), hyposmotic solution (red), hyperosmotic solution (orange), GSK101 (TRPV4 agonist; green) and capsaicin (TRPV1 agonist; gray). Activity from uninjected oocytes is shown in Ci. Inserts: Current activity at -85 mV obtained after exposure to -100mOsm, +100 mOsm, GSK101 or capsaicin were normalized to that obtained in control condition. N.S. = p > 0.05, *** = p< 0.001; one-way ANOVA, n = 12 oocytes.
	Figure 6 – The proline rich distant part of the N-terminus of TRPV4 is required for swelling-induced activation. (A) Schematics of the full-length TRPV4 N-terminus and deletants. (B) Micrographs of TRPV4 and the functioning deletant (‘Deletant I’) immunolabeled with phalloidin, anti-AQP4 and anti-TRPV4 antibodies confirming plasma membrane expression. (C) Summarized I/V curves from TRPV4 (left) and ‘Deletant I’ (right) with control solution (black), hyposmotic solution (red) GSK101 (green). (D) Current activity at -130 mV obtained in control solution, after exposure to -100mOsm or GSK101 in the full-length or deletants shown as bars. N.S. = p > 0.05, *** = p< 0.001; one-way ANOVA, n = 9 oocytes.
	Figure 7 – TRPV4 is activated by small osmotic gradients in Müller cells. (A) Representative raw trace of responses recorded in Fura-2AM loaded retinal ganglion cells. Application of osmotic gradients is indicated by the blue bar. Physiologically relevant osmotic gradients (-20 and -40 mOsm), and thus small volume changes do not elevate retinal ganglion cell calcium levels above spontaneous calcium spikes. Calcium levels were evoked upon application of a large gradient (-140 mOsm) serving as a positive control for responsiveness. Insert: Micrograph showing dissociated Müller and retinal ganglion cells. (B) Summary for results shown in (A). Shown are mean ± SD values of spontaneous manually-detected spikes (peak +/- 10 frames pre- and post-peak) before stimulation (Ctrl) and the change in [Ca2+]i measured as spikes detected above a threshold (stipled line) sat as spontaneous levels during stimulation (-20 or -40 mOsm), n = 19 cells. (C) Representative raw traces of responses recorded in Fura-2AM loaded Müller cells presented as in (A). Summary of results are shown in (D) and was analyzed and processed as in (B), n = 10-13 cells. (E) Representative raw trace of response recorded in Fura-2AM loaded Müller cells displaying reduced responsiveness in the presence of HC-06 with summary of results in (F), n = 19 cells. N.S. = p > 0.05 N.S. ; ** = p < 0.01; *** = p < 0.001; two-sample t-test.
	Figure 8 – PLA2 activation is required for swelling-induced TRPV4 activation in Müller cells. (A) Representative raw trace of response recorded in Fura-2AM loaded retinal ganglion cells. Activation of PLA2 (sc-3034) is indicated by the pink bar. (B) Summary for results shown in (A). Shown are mean ± SD values before PLA2 activation (Ctrl) and the change in [Ca2+]i during stimulation (sc-3034), n = 29 cells. (C) Representative raw trace of response recorded in Müller cells with summary of results shown in (D), n = 13 cells. (E) Representative raw trace of responses recorded Müller cells verifying their TRPV4 origin; The before evoked PLA2 activation induced responses are abolished in the presence of HC06. (F) Summary of results in (E), n = 10 cells. (G) Representative raw trace of response recorded in Müller cells after pretreatment with a PLA2 inhibitor (ONO-RS-089, indicated by the pink bar) prior to application of an osmotic gradient (-20 mOsm, indicated by the blue bar), and after ONO-RS-089 washout. (H) Summary of results from (G) presenting increase in swelling-induced calcium levels after washout of PLA2 blocker, n = 14 cells. N.S. = p > 0.05; ** = p < 0.01; *** = p < 0.001; two-sample t-test.

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