Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

Abstract

The present work investigates the effect of phosphatidylinositol-4,5-bisphosphate (PIP(2)) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co-immunoprecipitation methods. Inclusion of 100 microM diC8-PIP(2) in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)-evoked whole-cell cation currents and TRPC6 channel activity by over 90%. In inside-out patches diC8-PIP(2) also inhibited TRPC6 activity induced by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) with an IC(50) of 7.6 microM. Anti-PIP(2) antibodies potentiated Ang II- and OAG-evoked TRPC6 activity by about 2-fold. Depleters of tissue PIP(2) wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP(2) scavenger poly-L-lysine. Wortmannin reduced Ang II-evoked TRPC6 activity by over 75% but increased OAG-induced TRPC6 activity by over 50-fold. Co-immunoprecipitation studies demonstrated association between PIP(2) and TRPC6 proteins in tissue lysates. Pre-treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP(2). These results provide for the first time compelling evidence that constitutively produced PIP(2) exerts a powerful inhibitory action on native TRPC6 channels.

Figure 1. DiC8-PIP₂ inhibits Ang II-evoked whole-cell cation currents and single TRPC6 channel activity in rabbit mesenteric artery myocytes

Aa and b, inclusion of 100 μm diC8-PIP₂ in the patch pipette solution inhibits whole-cell cation currents evoked by 1 nm Ang II. Ac, mean I–V relationship showing diC8-PIP₂ inhibits Ang II-evoked cation currents at all potentials tested (n = at least 4 per point). Ba, 100 μm diC8-PIP₂ inhibited TRPC activity in an inside-out patch which was initially activated by 1 nm Ang II in a cell-attached patch (i/o, inside-out patch). Bb, 100 μm diC8-PIP₂ inhibited TRPC activity induced by 10 μm OAG in an inside-out patch. C, inhibitory action of diC8-PIP₂ on OAG-evoked TRPC6 activity in inside-out patches had an IC₅₀ value of 7.6 μm (n = at least 4 per point).

Figure 2. Anti-PIP₂ antibody potentiates TRPC6 channel activity

Aa, 1: 200 dilution of anti-PIP₂ antibodies transiently increased TRPC6 activity in an inside-out patch which was induced by 1 nm Ang II in cell-attached mode. Subsequently the Ang II-evoked response was inhibited in the presence of the anti-PIP₂ antibody. Ab, 1: 200 dilution of anti-PIP₂ antibodies produced a sustained increase of OAG-evoked TRPC6 activity in an inside-out patch. B, mean data of effect of anti-PIP₂ antibodies on Ang II- and OAG-induced TRPC6 activity (n = 6 for all conditions, **P < 0.01).

Figure 3. Agents that deplete PIP₂ evoke TRPC6 channel activity

A and B, respectively, 20 μm wortmannin and 100 μm LY294002 transiently activated channel activity in cell-attached patches (a) which had similar amplitude histograms (b) and unitary conductances and E_r values (c). C, 50 μg ml⁻¹ poly-l-lysine activated channel currents in an inside-out patch (a) which had a similar properties to those evoked by wortmannin and LY294002 (b and c).

Figure 4. Wortmannin-induced depletion of PIP₂ decreases Ang II-evoked and increases OAG-induced TRPC6 activity in cell-attached patches

A shows that Ang II-evoked TRPC6 activity (a) is decreased after pre-treatment with wortmannin (b). B illustrates that OAG-induced TRPC6 activity (a) is markedly increased following pre-treatment with wortmannin (b). C, mean data showing effect of wortmannin on Ang II- and OAG-induced TRPC6 activity (***P < 0.001). Da shows a co-immunoprecitation experiment in which mesenteric artery tissue lysates were immunoprecipitated (IP) with an anti-PIP₂ antibody and then Western blotted (WB) with an anti-TRPC6 antibody. In control conditions a band of ∼110 kDa was observed which was absent after pre-treatment of the tissue with 20 μm wortmannin, 1 nm Ang II and 10 μm OAG for 15 min. Note that similar results were obtained with both anti-TRPC6 antibodies (Alomone and Santa Cruz). Db, immunoblot with anti-PIP₂ antibody showing reduction of total tissue PIP₂ levels after incubation with 20 μm wortmannin for 15 min. Pre-treatment with 1 nm Ang II and 10 μm OAG for 15 min had little effect on total tissue PIP₂ levels. Dc, immunoblot with anti-TRPC6 antibody showing pre-treatment of tissue with 20 μm wortmannin, 1 nm Ang II and 10 μm OAG and for 15 min had no effect on expression of TRPC6 protein. Note that all immunoprecipiation and immunoblot experiments were from at least n = 3.