Fluorescence labeling strategies for the study of ion channel and receptor cell surface expression: A comprehensive toolkit for extracellular labeling of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. A critical aspect of measuring changes in ion channel expression is uniquely identifying ion channels located on the cell surface. To accomplish this goal we demonstrate two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb the function of the channel: 1) We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry compatible with a trans-cyclooctene coupled fluorescent dye. 2) By inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we incorporate a click-chemistry site for a chloroalkane-linked fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population, and we identified T468 as a rapid labeling site (∼5 minutes) based on functional as well as biochemical assays in HEK293T/17 cells. After several rounds of adapting the linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, our efforts led to a channel construct that robustly expressed as a fully functional TRPV1exCellHalo fusion with intact wild-type gating properties. The TRPV1exCellHalo construct was used in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The success of these extracellular label TRPV1 (exCellTRPV1) constructs as tools to track surface expression of the channel will shed significant light on the mechanisms regulating expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

Conspecific interactions predict social transmission of fear in female rats.

Social transmission of fear occurs in a subset of individuals, where an Observer displays a fear response to a previously neutral stimulus after witnessing or interacting with a conspecific Demonstrator during memory retrieval. The conditions under which fear can be acquired socially in rats have received attention in recent years, and suggest that social factors modulate social transmission of information. We previously found that one such factor, social rank, impacts fear conditioning by proxy in male rats. Here, we aimed to investigate whether social roles as determined by nape contacts in females, might also have an influence on social transmission of fear. In-line with previous findings in males, we found that social interactions in the home cage can provide insight into the social relationship between female rats and that these relationships predict the degree of fear acquired by-proxy. These results suggest that play behavior affects the social transfer/transmission of information in female rats.