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

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 μ m 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 μ m . 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.