FIGURE 5.

Defects associated with the SS mutation are not caused by decreased half-life in the plasma membrane or alterations in channel gating. a, confocal microscopy of fixed HeLa cells transfected with either full-length WT or SS TRPC3 (green) and 10 kDa Texas Red-tagged dextran. Top, arrows show areas of co-localization. WT TRPC3 co-localizes with internalized dextran, while only a fraction of dextran-containing vesicles are positive for SS TRPC3. b, Western blot of biotinylated HEK293 cells (left) and loads (right) transfected with either Myc-tagged WT or SS TRPC3 alone, or coexpressed with a dominant-negative dynamin mutant (DIIKA), which blocks endocytosis. These preparations were treated with or without 100 μm CCH. Input lanes, 30 μg. Anti-HO2 blot serves as an intracellular negative control for biotinylation. c, Fura2AM measurements were made in HEK293 cells non-transfected (con, black) or transfected (red) with either WT or SS TRPC3 alone, or SS TRPC3 + dominant-negative DIIKA. Cells were acclimated first in nominally Ca²⁺-free medium (A, arrow), Ca²⁺ pools were released by 100 μm CCH in nominally Ca²⁺-free medium (B, arrow) followed by replacement with CCH and 1 mm Ba²⁺-containing media (C, arrow).