Abstract
Purpose:
Current analgesic treatment for corneal damage or following ophthalmic surgery falls short of desired goals. One promising approach is the temporary deactivation of TRPV1 (transient receptor potential vanilloid 1) expressing nociceptors by administration of the TRPV1 agonist resiniferatoxin. Here we consider the molecular pathways of TRPV1 regulation. Inflammatory signals increase the excitability of TRPV1-expressing nociceptors, at least in part, by increasing the number TRPV1 channels in the plasma membrane (PM) of nociceptors. However, the dynamics of TRPV1 in the PM have not been studied. We hypothesize that TRPV1 properties are heterogeneous within a cell and that channels are actively regulated as a function of local activity.
Methods:
We used total internal reflection fluorescence (TIRF) microscopy to explore the dynamics of single TRPV1 molecules in living cells. Combining TIRF microscopy with whole-cell patch clamp of isolated mouse sensory neurons and HEK293T/17 cells allowed us to image both the localization of single TRPV1 molecules within cells (TRPV1-eGFP) and their activity (capsaicin-activated fluorescent “sparklets” at site of Ca2+ influx).
Results:
A number of surprising findings came out of our single-molecule experiments: (1) TRPV1 channels in isolated sensory neurons and in cultured cells are mobile, with a range of Deff from 0.01 to 0.2 µm2/s; (2) a given Ca2+ sparklet arises from exactly one channel, with two-state fluorescence reflecting capsaicin-activated gating; (3) photobleaching and fluorescence intensity analysis indicate that TRPV1 channels do not cluster in the PM; and (4) the lateral mobility of TRPV1 in the plasma membrane decreased as a function of its open duration.
Conclusions:
Our data show that TRPV1 activates without forming a higher order oligomer and that a subset of channels exhibit lateral mobility as they gate. Although the mechanism by which TRPV1 activity and TRPV1 mobility are coupled and the role of mobility changes in cell signaling remain to be determined, our data demonstrate the power of single-molecule measurements to reveal aspects of signaling not observable in macroscopic experiments. Given previous work indicating that TRPV1 is part of a signaling complex including the TrkA receptor for nerve growth factor and PI3K, our data suggest that dynamic localization of these complexes in response to TRPV1 agonists may constitute a new form of regulation of local signaling.
Keywords: 480 cornea: basic science •
569 ion channels •
715 signal transduction: pharmacology/physiology