Fig. 5.
TRPM4 translocation and fusion with the plasma membrane. (A) Cellular localization of flag-TRPM4, in resting flag-TRPM4-TrexHEK293 (left panels), and l μM ionomycin treated cells (right panels), stained for flag-TRPM4 expression (red) with 2.5 mg/ml mouse anti-Flag primary antibody (Sigma), and visualized using an Alexa-568 conjugated anti-mouse secondary antibody (Invitrogen). Cell bodies were delineated using 1 mM Cell Tracker (green) prior to fixing. Confocal Z-series were taken using a BioRad 1024 inverted confocal laser microscope, with krypton/argon laser. Top panels are projected Z-stacked images taken at 0.65 mm increments through the cell, bottom panels are z-axis interpolated x-axis sections through the cell. Note the initial punctate localization of TRPM4 and shift of fluorescence to a plasma membrane localization following ionomycin treatment. (B) Average inward currents from ΔN-TRPM4 expressing cells (n = 14) and non-tetracycline induced control cells (n = 7) at −80 mV with [Ca2+]i buffered at 1 μM (mean ± S.E.M.). (C) Normalized capacitance changes from ΔN-TRPM4 expressing and control cells. Inhibition of TRPM4 currents by a dominant negative effect is clearly visible, but does not alter exocytosis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)