Figure 6. TrpC3 channel localization is distinct from that of CaV1.2.
(A–B) Epifluorescence microscopy images of fixed myocytes expressing TrpC3 C-terminally tagged with YFP (TrpC3-YFP). Cells were stimulated for 48 hours with PE to induce hypertrophy. The majority of cells display a punctate and perinuclear pattern of TrpC3-YFP (example shown in A, left panel). Co-staining with anti-α-actinin antibodies (A, right panel) reveals the localization of the Z-lines, which are closely apposed to the t-tubules. Magnifying a merge of the TrpC3-YFP and anti-α-actinin images (A, inset) shows little overlap between TrpC3-YFP puncta and the Z-lines. In some transfected myocytes, TrpC3-YFP puncta are completely excluded from the Z-lines (example shown in B, inset). (C) TrpC3-YFP expressed in a serum-starved, un-hypertrophied myocyte (left panel) is also distributed in puncta. The same punctate pattern is also observed in hypertrophied myocytes (right panel) expressing TrpC3 with an N-terminal YFP tag (YFP-TrpC3). (D) Live myocytes co-transfected with CFP-Myr (a marker of the plasma membrane) and TrpC3-YFP were imaged by epifluorecence and total internal reflection (TIRF) microscopy. CFP-Myr imaged by epifluorescence (left panel) illustrates the extent of the plasma membrane. A TIRF image of TrpC3-YFP (right panel) indicates that, in live myocytes, tagged TrpC3 is found in puncta within 200 nm of the cell surface.