Abstract
Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P2 in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P2 was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P2 in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P2 in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P2 whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P2 must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P2 may resolve the long standing controversy about whether PI(4,5)P2 is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.
Highlights
Whether phosphoinositide 4,5-bisphosphate (PI[4,5]P2) activates or inhibits TRPV1 is controversial
We show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI[4,5]P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in
The exogenous agonist capsaicin freely equilibrates across the membrane so that it is effective when applied to the extracellular face of an outside-out patch as it is when applied to the intracellular face of an inside-out patch [23]
Summary
Whether phosphoinositide 4,5-bisphosphate (PI[4,5]P2) activates or inhibits TRPV1 is controversial. Membrane Asymmetry Required for PIP2 Activation of TRPV1 cell - asymmetric liposomes - symmetric extracellular leaflet no PI[4,5]P2 inside-out patch intracellular leaflet. PI[4,5]P2 symmetrically localized to both leaflets inhibited TRPV1, this required a very high concentration of PI[4,5]P2 in the extracellular leaflet These findings may explain the anomalous inhibition of reconstituted TRPV1 channels by PI[4,5]P2 in symmetric liposome patches and emphasize the importance of membrane asymmetry when using reduced systems for studies of membrane proteins
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