Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4

Abstract

TRPV4 is a Ca(2+)- and Mg(2+)-permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca(2+)-dependent signal transduction in several tissues, including brain and vascular endothelium. TRPV4-activating stimuli include osmotic cell swelling, heat, phorbol ester compounds, and 5',6'-epoxyeicosatrienoic acid, a cytochrome p450 epoxygenase metabolite of arachidonic acid (AA). It is presently unknown how these distinct activators converge on opening of the channel. Here, we demonstrate that blockers of phospholipase A(2) (PLA(2)) and cytochrome p450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4alpha-phorbol 12,13-didecanoate. Mutating a tyrosine residue (Tyr-555) in the N-terminal part of the third transmembrane domain to an alanine strongly impairs activation of TRPV4 by 4alpha-phorbol 12,13-didecanoate and heat but has no effect on activation by cell swelling or AA. We conclude that TRPV4-activating stimuli promote channel opening by means of distinct pathways. Cell swelling activates TRPV4 by means of the PLA(2)-dependent formation of AA, and its subsequent metabolization to 5',6'-epoxyeicosatrienoic acid by means of a cytochrome p450 epoxygenase-dependent pathway. Phorbol esters and heat operate by means of a distinct, PLA(2)- and cytochrome p450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain.

Fig. 1.

Tyr-253 is not required for the hypotonicity-induced activation of TRPV4. (A) Effect of stimulation with 25% hypotonic solution on [Ca²⁺]_i in nontransfected (NT) HEK cells (dashed line) and cells transfected with the Y253F mutant (solid line). (B, left) Average [Ca²⁺]_i increases induced by hypotonic stimulation in NT cells (n = 31), WT TRPV4-transfected cells (HTS, n = 32), and TRPV4-transfected cells treated with the tyrosine kinase inhibitors PP1 (10 μM; n = 22) or genistein (Gen; 100 μM; n = 23). (B, right) Average [Ca²⁺]_i increases in Y253F-transfected cells induced by hypotonic cell swelling (HTS; n = 22), 1 μM4α-PDD (4αPDD; n = 18), 10 μMAA(n = 21), and heating to 42°C (Heat; n = 22). For every condition, data from at least three independent experiments were pooled.

Fig. 2.

Effect of the PLA₂-inhibitor pBPB on [Ca²⁺]_i in HEK cells expressing WT TRPV4. (A) Effect of stimulation with 25% hypotonic solution on [Ca²⁺]_i in nontransfected (NT) HEK cells (dashed line), TRPV4-transfected cells (solid line), and TRPV4-transfected cells treated with 100 μM pBPB (dotted line). Note the significantly higher basal [Ca²⁺]_i levels in TRPV4-transfected cells (see Fig. 7). (B) Dose–response curve for the inhibition of the hypotonicity-induced [Ca²⁺]_i increase by pBPB (○) and ACA (•). (C and D) Same as A, except that stimulation was with 1 μM 4α-PDD (C) or 10 μM AA (D). (E) Average [Ca²⁺]_i increases in response to 1 μM4α-PDD, 25% hypotonic solution, 10 μM AA, and heating to 42°C in NT cells and TRPV4-transfected cells treated with four different PLA₂ inhibitors (100 μM pBPB, 10 μM OBAA, 20 μM AACOCF₃, and 20 μM ACA, respectively). For every condition, n ≥ 16 in at least three independent experiments. *, P < 0.05, compared with nontreated TRPV4-expressing cells.

Fig. 3.

Effect of PLA₂ inhibitor pBPB on whole-cell currents in HEK cells expressing WT TRPV4. (A) Time course of whole-cell currents at -80 and 80 mV in a TRPV4-transfected HEK cell, showing current activation on stimulation with hypotonic solution (HTS). (B) Current–voltage relations obtained at the time points indicated in A. (C and D) Same as A and B, except that treatment was with 100 μM pBPB. Note that no current activation was observed. (E and F) Same as in A and B, except that TRPV4-transfected HEK cells were stimulated with 1 μM4α-PDD. (G and H) Same as in E and F, showing the lack of effect of pBPB on 4α-PDD-induced TRPV4 activation.

Fig. 4.

Blockers of cytochrome P450 epoxygenase inhibit the hypotonicity-induced activation of TRPV4. (A) Effect of stimulation with 25% hypotonic solution on [Ca²⁺]_i in nontreated TRPV4-transfected HEK cells (solid line) and TRPV4-transfected HEK cells treated with miconazole (10 μM; dashed line). (B) Effect of miconazole (10 μM) and 17-ODYA (10 μM) on the elevation of [Ca²⁺]_i induced by hypotonic stimuli (filled bars) and heating to 42°C (open bars). For every condition, n ≥ 22 in at least three independent experiments. *, Significant differences, compared with nontreated TRPV4-expressing cells.

Fig. 5.

Effect of various activation stimuli on [Ca²⁺]_i in HEK cells expressing the TRPV4 YS mutant (Y555A/S556A). (A–D) Time course of Ca²⁺ concentration in nontransfected cells (dashed line) and cells expressing the YS mutant (solid line) on stimulation with 1 μM4α-PDD (n = 65) (A), heating to 42°C(n = 34) (B), 25% hypotonic solution (n = 27) (C), or 10 μM AA (n = 24) (D). (E) Average [Ca²⁺]_i increases in nontransfected HEK cells and HEK cells transfected with TRPV4 WT, Y555A, Y555F, S556A, Y555A/S556A, and Y555F/S556A in response to 1 μM 4α-PDD, 10 μM AA, 25% hypotonic solution, and heating to 42°C, respectively. For every condition, n ≥ 17 in at least three independent experiments. *, Significant differences, compared with cells expressing WT TRPV4.

Fig. 6.

Effect of the YS mutation on activation of TRPV4 currents by agonist stimulation and hypotonic swelling. (A–H) Time course of whole-cell currents at -80 and +80 mV (A, C, E, and G) and current–voltage relations obtained at the indicated time points (B, D, F, and H) in HEK cells expressing the Y555A/S556A mutant, showing the lack of response to 4α-PDD (A and B) and heating to 42°C (C and D) and current activation on stimulation with 25% hypotonic solution (E and F) and 10 μMAA(G and H). (I) Maximal increases in inward-current density at -80 mV on stimulation with 1 μM 4α-PDD in nontransfected cells (n = 7), cells expressing WT TRPV4 (n = 6), and the F548A (n = 5), Y555A/S556A (YS/AA; n = 11), and Y552A (n = 6) mutants. (J) Comparison of the average inward current activated by 25% hypotonicity, 10 μM AA, and heating to 42°C in nontransfected cells and cells expressing WT and mutant TRPV4 (n = 5–13). *, Significant differences, compared with cells expressing WT TRPV4.