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. 2012 Apr 6;287(23):19462–19471. doi: 10.1074/jbc.M111.289751

FIGURE 4.

FIGURE 4.

Agonist-induced TRPV1 is internalized through a clathrin- and dynamin-independent pathway. A, upper panel, representative Ca2+ imaging traces in TRPV1+ cells in the absence (black) or presence (dotted line) of a hyperosmotic sucrose solution. Two 1-min pH 5.5 pulses, pH1 and pH2, were applied interspersed by a 20-min incubation period in the absence (vehicle (V)) or presence of 1 μm capsaicin. Although a significant desensitization (expressed as RpH2/pH1) was evident under normosmotic conditions (−Sucr), the constant presence of hyperosmotic solution (+Sucr) significantly reduced capsaicin-induced TRPV1 desensitization. Lower panel, the time course of desensitization, which reaches its maximum after 20 min. ***, p < 0.001. ns, not significant. B, biotinylation of cell surface proteins in the presence of hyperosmotic 250 mm sucrose solution (600 mosm)) reveals a block of capsaicin-induced TRPV1 internalization. n = 3. ***, p < 0.001. IB, representative blot; Memb, membrane. C, measurement of Ca2+ influx in TRPV1-WT, using the same protocol as in A, in the presence of 50 μm Chpz (solid line) as compared with 0.1% DMSO used as Chpz vehicle (dotted line). Rel. Response, relative response. D, Ca2+ influx in TRPV1-WT, using the same protocol as in A, in cells co-transfected with GFP-fused WT dynamin (dotted trace) or the dominant-negative mutant GFP-Dyn-K44A (solid trace). Capsaicin-induced internalization occurs through a dynamin-independent process. E, representative images of TRPV1+ cells expressing red fluorescent fusion protein-mouse clathrin light A (mLCA-RED). TRPV1 was surface-labeled (green) and then allowed to internalize upon incubation with 1 μm capsaicin for 20 min in the presence of hyperosmotic solution (bottom) as compared with normosmotic conditions (top) and then fixed and visualized. Bar, 10 μm. F, immunocytochemistry of extracellularly labeled TRPV1 (red) internalized in the presence of 1 μm capsaicin for 20 min in the presence of GFP-fused WT dynamin (left) or the dominant-negative dynamin mutant GFP-Dyn-K44A (right) shows that capsaicin-induced TRPV1 internalization occurs through a dynamin-independent process.