Cell Unroofing to Study the PI3K-TRPV1 Interaction

Abstract

Transient receptor potential vanilloid 1 (TRPV1) channels play an essential role in the sensitized response to painful stimuli that occurs in response to inflammatory mediators including nerve growth factor (NGF). Sensitization involves NGF activation of phosphoinositide 3-kinase (PI3K), which increases the concentration of the signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 then triggers exocytosis of TRPV1-containing vesicles, increasing the number of TRPV1 channels on the plasma membrane. We have previously shown that TRPV1 and PI3K interact directly through the channel's N-terminus and PI3K's p85 subunit. However, little is known about the dynamics behind the interaction and its consequences. Cell unroofing is an effective method for accessing biological membranes from the inside. In cell unroofing, a probe sonicator is mounted on the microscope stage and positioned within about 1 mm of adherent cells. A single pulse of the sonicator shears off the top of the cells, causing loss of organelles and cytosolic components, and leaving a plasma membrane sheet with its proteins and lipids intact. Because of the near-instantaneous disruption of cellular contents, this system is ideal for measuring dissociation rate of membrane-associated proteins. By labeling proteins of interest with fluorescent tags, we can measure the time course of the decay in fluorescence that occurs upon unroofing. We used PI3K fused to YFP and compared the dissociation rates in cells without TRPV1 to those that were transiently transfected with TRPV1. We quantified the fluorescent signals and obtained the dissociation rates (koff) by fitting the data with an exponential decay model. These studies revealed that PI3K dissociation from the plasma membrane was slower in TRPV1-expressing cells than in those not expressing TRPV1. We conclude that PI3K interacts with TRPV1 in resting cells. Whether the interaction is altered in NGF-stimulated cells is still an open question.