Volume 15, Issue 4, Pages 635-646 (August 2004) VAMP2-Dependent Exocytosis Regulates Plasma Membrane Insertion of TRPC3 Channels and Contributes to Agonist-Stimulated Ca2+ Influx  Brij B. Singh, Timothy P. Lockwich, Bidhan C. Bandyopadhyay, Xibao Liu, Sunitha Bollimuntha, So-ching Brazer, Christian Combs, Sunit Das, A.G.Miriam Leenders, Zu-Hang Sheng, Mark A. Knepper, Suresh V. Ambudkar, Indu S. Ambudkar  Molecular Cell  Volume 15, Issue 4, Pages 635-646 (August 2004) DOI: 10.1016/j.molcel.2004.07.010

Figure 1 Interaction of TRPC3 with SNARE Proteins (A) Yeast two hybrid interaction of NTRPC3 or CTRPC3 with VAMP2 and αSNAP. Nd, not detected. Crude membranes (CM) prepared from either HEK-293 cells (B) or HSG cells (C) stably expressing either HA-TRPC3 or FLAG-TRPC3 (Biv) were solubilized and immunoprecipitation (IP) was performed as described in Experimental Procedures. Proteins in the IP were detected by SDS-PAGE and immunoblotting (IB). Antibodies used for IP and IB are indicated in the figure. (Bvi) Control IPs using non-transfected 293 cells or HA-TRPC3-293 cells (IgG; control rat IgG, similar results were obtained with rabbit IgG, not shown). Inputs shown here and in subsequent figures were 1/10–1/20 of the protein used for IP. Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)

Figure 2 Colocalization of HA-TRPC3 with Endogenous SNARE Proteins in HEK-293 Cells Anti-HA antibody and rhodamine-conjugated secondary antibody were used to detect HA-TRPC3 (i–vi, red images) stably expressed in HEK-293 cells. Other antibodies were as follows: (i) anti-VAMP2, (ii) anti-αSNAP, (iii) anti-syntaxin3, (iv) anti-SNAP23, (v) anti-NSF, (vi) anti-VAMP3. All of these proteins were detected using FITC-conjugated secondary antibody (green images). Overlay of images in each case is shown in the right panels (yellow signal). The box shows an enlarged image of TRPC3 from the area in (ii) indicated by white rectangle. Arrows in the images show the protein localization. Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)

Figure 3 Colocalization of Endogenous TRPC3 with SNARE Proteins in Cultured Rat Hippocampal Neurons Anti-TRPC3 antibody (green signal in [A], [B], [C], and [D]), anti-αSNAP antibody ([A], red signal); anti-syntaxin 3 ([B], red signal); anti-synaptophysin ([C], red signal), and anti-VAMP2 ([D], red signal) were used to detect the proteins. Arrows in (C) and (D) show synaptophysin and VAMP2 signals in the synaptic terminals. (C) and D also show DIC images of the neurons. Overlay of the images (right panels, yellow signal) shows that TRPC3 is not concentrated in the synaptic terminals. High-resolution images of TRPC3-synaptophysin and TRPC3-VAMP2 in (C) and (D), respectively show overlay and DIC images. Immunoprecipitation of TRPC3 with VAMP-2 (E) and syntaxin3 (F) from solubilized rat brain CM. (G) Control IP using rabbit IgG. Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)

Figure 4 Exocytotic Trafficking of TRPC3 to the Plasma Membrane (A) Localization of GFP-TRPC3 in HEK-293 cells. Arrow indicates mobile intracellular vesicular structures containing GFP-TRPC3. (B) FRAP measurements in GFP-TRPC3-expressing cells: control cells and cells treated with TeNT (100 nM, 6 hr), Brefeldin A (BFA, 10 μg/ml, 1 hr), or loaded with BAPTA-am (incubated with 50 μM for 30 min). Arrows indicate the photobleached area. (C) Representative time-course of recovery in control cells. (D) t1/2 of fluorescence recovery calculated from graphs such as that shown in (C). *, a statistically significant difference as compared to other values given in the table (p < 0.05, Student's t test). Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)

Figure 5 Tetanus Toxin Treatment Decreases Plasma Membrane Levels of TRPC3 (A) Localization of TRPC3 and VAMP2 in HA-TRPC3-expressing HEK-293 cells (control, TeNT, or TeNT-treated, +TeNT). HA-TRPC3 was detected using anti-HA antibody and rhodamine-conjugated secondary antibody (red signal, left panels), VAMP2 was detected using anti-VAMP2 antibody and FITC-conjugated secondary antibody (green signal, middle panels), overlay images (yellow signal, right panels). (B) Top panel: Western blot showing VAMP2 in TeNT-treated cells. Lower panel: Surface expression of exo-HA-TRPC3 in non-permeabilized untreated 293 cells cells (left image) and TeNT-treated cells (right image). (C) Representative FACS analysis of exo-HA-TRPC3 in HEK-293 cells (data from a representative experiment are shown) detected using anti-HA and FITC-conjugated secondary antibodies. The upper-right and -left panels show the side and forward light scatter of control (left) and TeNT-treated (right) cells. Lower-left panel, the light scatter profile of BFA- treated cells (scatter plot of BAPTA-treated cells were similar to these and is not shown). Lower-right panel, histogram (blue, control non-transfected cells; orange, TRPC3 cells; pink, TRPC3+TeNT; green, TRPC3+BFA. TRPC3+BAPTA is not shown) of the fluorescence intensities. (D) Relative levels of surface TRPC3 calculated from the histogram data. *, a significant (p < 0.03, n = 4 for each) difference compared to unmarked values. Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)

Figure 6 Tetanus Toxin and Brefeldin A Decrease CCh-and OAG Stimulation of TRPC3 Activity Ca2+ or Sr2+ influx were measured in OAG and CCh-stimulated HA-TRPC3 expressing control cells (TRPC3) and cells treated with TeNT or BFA. (A, C, E, and G): fluorescence traces in either OAG- (A and E) or CCh- (C and G) treated cells. (B, D, F, and H) average data. **, values that are significantly different from that of the respective control condition (p < 0.02, n is indicated in each case). (I, J, and K) Effect of TeNT on thapsigargin (Tg)-stimulated Ca2+ mobilization. Internal Ca2+ release, Ca2+ influx, and basal Ca2+ influx were not altered by TeNT treatment. (Li) Presence of endogenous TRPC3 in HEK-293 cells: crude membrane fraction from FLAG-TRPC3-expressing (lane 1) or non-transfected (lane 2) cells. Anti-TRPC3 antibody detects the same protein in both samples. (Lii) Coimmunoprecipitation of endogenous TRPC3 and VAMP2. (M, N, and O) Effect of TeNT on CCh-stimulated sustained Ca2+ increase in non-transfected HEK-293 cells (shown by arrow). Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)

Figure 7 CCh Regulates Surface Expression of TRPC3 (A) Surface biotinylation of FLAG-TRPC3- expressing HEK-293 cells (see Experimental Procedure for details). Cells, either unstimulated (-CCh) or stimulated (+CCh, 100 μM CCh for 1 min in Ca2+-free medium) were lysed and equal amounts of protein were used for affinity pull-down with either Neutr-Avidin-linked beads (A, B, C, and G–I) or anti-FLAG (C). Imunoblotting was done with Neutr-Avidin (to show the biotinylation pattern, [A]) or anti-FLAG ([B, C, and E–J], other antibodies used for IB are indicated). (D) Effect of CCh on surface expression of endogenous TRPC3 (using non-transfected 293 cells), Na+/K+ATPase, and PMCA. (E–G) Cells were treated with BAPTA or TeNT and then with carbachol (+CCh) or vehicle (-CCh). Upper panels, inputs (1/20 of IP); middle panels, avidin pull-down probed with anti-FLAG antibody; and lower panels, quantitation of the bands (Image Quant, Molecular Devices) detected in the Western blots (number of samples in [E], [F], and [G] were 12, 5, and 7, respectively). In each group, the +CCh condition is represented relative to the –CCh condition. *, values that are significantly different from other values but not from each other. **, a value that is significantly different from the unmarked values as well as those marked *. Hatched bars in (F) and (G) indicate that these values were lower compared to the -CCh values in the control group. (H) Effect of thapsigargin (3 min, Ca2+-free medium) on surface expression of FLAG-TRPC3. I. Non-transfected 293 cells were treated with TeNT and then with CCh as described above. Endogenous TRPC3 in the input samples as well as avidin-IPs are shown. (J) Avidin pull down from non-biotinylated and biotinylated FLAG-TRPC3 expressing cells (unstimulated). Blots were probes with either anti-FLAG (upper panel) or anti-TRPC3 (lower panel). Input level of TRPC3 in non-biotinylated and biotinylated samples were similar when probed with anti-FLAG or anti-TRPC3 (not shown). Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)

Figure 8 Surface Expression of TRPC3 In resting cells intracellular vesicles containing SNARE proteins and TRPC3 traffic to the plasma membrane and interact with it via SNAREs and other as yet unknown docking and scaffolding proteins (not indicated in the figure). Vesicle fusion results in surface expression of TRPC3. Cytosolic Ca2+ as well as membrane PIP2 (via promoting internalization) could control this trafficking. In stimulated cells, PIP2 hydrolysis could regulate surface expression of TRPC3 by regulating this trafficking process (increase fusion or decrease internalization). Further, channel activation could be induced by the local action of DAG. Alternatively, active channels could be inserted (this possibility needs to be assessed). Whether depletion of internal Ca2+ stores and/or interaction with ER proteins such as IP3R exert additional effects on the trafficking and function of TRPC3 is not yet clear. Our data suggest that CCh-stimulated increase in surface expression of TRPC3 depends on VAMP2-dependent exocytosis but not on [Ca2+]i increase. Molecular Cell 2004 15, 635-646DOI: (10.1016/j.molcel.2004.07.010)