Optical Recording of Single-Channel TRPV1 Activity and Mobility in Isolated Dorsal Root Ganglion (DRG) Neurons and Cultured Mammalian Cells

Abstract

TRPV1 is a calcium-permeable non-selective cation channel recognized for its sensitivity to heat, low pH, oxidation and numerous endogenous agonists. To date there is very little known about the activity of the individual channel in the intact cell. In order to explore TRPV1 in this context we employed total internal reflection fluorescence (TIRF) microscopy to observe the open states of the channel as a fluorescent “sparklet” at the site where calcium influx occurs. Preliminary investigations into the sparklet activity elicited by capsaicin in DRG neurons indicate that TRPV1 is free to move laterally in the plasma membrane while conducting calcium into the cytosol. Importantly, two-state fluorescent sparklets indicate that channels function independently in vivo. In HEK293T/17 cells transiently transfected with TRPV1-eGFP we simultaneously imaged mobile channels by their eGFP label and their capsaicin-activated sparklets. We implemented both the eGFP photobleaching subunit counting strategy as well as sparklet intensity analysis of TRPV1 sites. Consistent with results in DRG neurons, we found that individual channels, rather than assemblies of channels, were present and functional in the plasma membrane. Further analysis of the subset of mobile sparklets revealed that the mobility of TRPV1 sparklets steadily decreased with its open duration. Activity dependence of TRPV1 mobility may represent a new form of channel regulation in which its function becomes spatially compartmentalized.