Membrane Asymmetry: Key to Phosphoinositide (4,5)-Bisphosphate Regulation of TRPV1

Abstract

The signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) is known to regulate the activity of many ion channels. For TRPV1 there is general agreement that PI(4,5)P2 is important for regulating activity, but whether as an activator or inhibitor remains a topic of debate. Here, we show that PI(4,5)P2 can activate or inhibit TRPV1 activity, depending on whether it is asymmetrically localized to the intracellular leaflet of the plasma membrane or whether it is present in both leaflets. When PI(4,5)P2 was localized to its physiological site, the intracellular leaflet of the plasma membrane, we confirmed that it activated TRPV1. In contrast, we observed an inhibition of TRPV1 activity when we introduced dic8-PI(4,5)P2 or BODIPY-labeled PI(4,5)P2 to the extracellular leaflet of outside-out excised patches so that, in combination with endogenous PI(4,5)P2 in the intracellular leaflet, localization of PI(4,5)P2 was no longer asymmetric. Interestingly, calibrations of the mole fraction of PI(4,5)P2 with isothermal titration calorimetry showed that much higher concentrations of PI(4,5)P2 in the extracellular leaflet were required for inhibition (≥3 mole%) compared to the concentrations of PI(4,5)P2 in the intracellular leaflet that produced activation (<0.1 mole%). In patch-clamp fluorometry experiments, application of BODIPY-labeled PI(4,5)P2 onto outside-out excised patches revealed that PI(4,5)P2 incorporation into the membrane is needed for its inhibitory effect. The opposite effects of PI(4,5)P2 on TRPV1 activity may reconcile the activation of TRPV1 by PI(4,5)P2 in physiological conditions with inhibition of TRPV1 by PI(4,5)P2 reported in a reconstituted system(Cao et al., 2013). Our results also underscore the importance of membrane asymmetry, especially in functional studies of membrane proteins.