Fig 2: NCAM140-ICD and NCAM180-ICD directly bind to the N-terminal ICDs of TRPC1, -4, and -5. Recombinant N-terminal ICDs of TRPC1 (TRPC1-ICDNT) (a,d), TRPC4 (TRPC4-ICDNT) (b,e), and TRPC5 (TRPC5-ICDNT) (c,f) were substrate-coated and incubated with increasing concentrations of recombinant NCAM140-ICD (a–c), NCAM180-ICD (a–c), CHL1-ICD (a–c), or NCAM140-ICD with a deletion of the N-terminal (NCAM140?N), middle (NCAM140?M), or C-terminal (NCAM140?C) parts (d–f). Binding was determined by ELISA using NCAM antibody P61 (a–c), CHL1 antibody (a–c), and NCAM antibody GTX133217 (d–f). Mean values ± SEM from 3 independent experiments carried out in triplicates are shown.

Fig 3: H1 receptor, Vglut2, TRPC1, TRPC7 and TRPC5 transcripts are present in MnPO neurons.Representative gels illustrating the expression of H1 receptor, Vglut2, TRPC1, TRPC7 and TRPC5 transcripts in 10 (1–10) acutely dissociated MnPO neurons. The expected sizes of the PCR products are (in base pairs) 286, 393, 208, 245 and 243, respectively. Negative (-) control was amplified from a harvested cell without reverse-transcription, and positive control (+) was amplified using 1 ng of hypothalamic mRNA. Cells 4 and 6–10 were excited by histamine (50 µM). Cells 1–3 and 5 were not affected by histamine.

Fig 4: TRPC5 induces autophagy via the CaMKKß/AMPKa/mTOR pathway in response to chemotherapy. (A and B) Treatment of indicated cells with ADM for 48 h increased the basal [Ca2+]i and siTRPC5 blocked the ADM-induced increase in [Ca2+]i. (C) Exposure of indicated cells to ADM for 48 h was followed by analysis of the protein levels of p-CaMKKß, CaMKKß, p-AMPKa, AMPKa, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB by western blot. (D) The effect of TRPC5 silencing on the protein levels of p-CaMKKß, CaMKKß, p-AMPKa, AMPKa, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB in indicated cells exposed to ADM. (E) The effect of 20 µmol/L BAPTA/AM on the protein levels of p-CaMKKß, CaMKKß, p-AMPKa, AMPKa, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. (F) The effect of CaMKKß silencing on the protein of CaMKKß, p-AMPKa, AMPKa, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells treated with ADM. (G) The effect of AMPKa silencing on the protein levels of AMPKa, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. (H and I) The effect of BAPTA/AM, siCaMKKß and siAMPKa on ADM induced LC3 puncta formation in indicated cells. (J and K) The effect of 10 µmol/L STO-609 and 5 µmol/L compound C on ADM induced LC3 puncta formation in indicated cells. (L) The effect of BAPTA/AM, STO-609 and compound C on accumulation of LC3-II in MCF-7/ADM cells. Values are mean ± SEM of 3 to 6 experiments. *p < 0.05, **p < 0.01, compared to siCTL or vehicle.

Fig 5: NCAM and TRPC1, -4, and -5 interact via their ICDs. (a,b) A brain detergent extract was used for immunoprecipitation (IP) with TRPC1 antibody H-105, TRPC4/5 antibody H-80 (4/5), TRPC3/6/7 antibody H-100 (3/6/7), and nonimmune control antibody (Ig). The brain detergent extract (input) and the immunoprecipitates were subjected to Western blot (WB) analysis with NCAM antibody 5B8 (NCAM140/180) (a), NCAM antibody D3 (NCAM180), or TRPC3/6/7 antibody H-100 (3/6/7) (b). (c) Detergent extracts of membrane-enriched brain fractions (input) and TRPC1 antibody ACC-010 (1), TRPC4 antibody ACC-018 (4), TRPC5 antibody ACC-020 (5), and a nonimmune control antibody (Ig) were used for immunoprecipitation, and the NCAM antibody GTX133217 against the C-terminus (NCAM-CT), a chicken antibody against the extracellular domains of NCAM (NCAM-ECD), and PSA antibody 735 (PSA) were used for Western blot analysis. (a–c) Arrowheads indicate bands representing NCAM140, NCAM180, and polysialylated NCAM140 and NCAM180 (PSA-NCAM), and the arrow indicates TRPC3, -6, or -7 bands. (d) Immunoprecipitation was carried out with brain detergent extracts (input) from wild-type (+/+) and NCAM-deficient (-/-) mice using NCAM antibody H28 and followed by Western blot analysis with TRPC1 antibody E-6 (1), TRPC4 antibody N77/15 (4), TRPC5 antibody N67/15 (5), and TRPC3/6/7 antibody H-100 (3/6/7). Arrows indicate TRPC1, -4, or -5 bands. (e,f) NCAM antibody 5B8 (NCAM140/180), D3 (NCAM180) (e), and GTX133217 (NCAM-CT) (f) were used for Western blot analysis of precipitates from pull-down experiments using detergent brain extracts (input) (e) or a membrane-enriched fraction (input) (f) and beads only (ctrl) or beads with GST, GST-tagged N-terminal (1N), or C-terminal (1C) ICDs of TRPC1 or GST-tagged N-terminal ICDs of TRPC4 (4N) or TRPC5 (5N). Arrowheads indicate NCAM180 and PSA-NCAM bands. (g–i) TRPC1 antibody E-6 (g,h) or TRPC3/6/7 antibody H-100 (i) were used for Western blot analysis of precipitates from pull-down experiments with detergent brain extracts and His-tagged ICDs of NCAM140 (140) (g–i), NCAM180 (180) (g,i), L1 (h,i), or NCAM140 with deletion of the N-terminal (?N), middle (?M), or C-terminal (?C) part (h). Arrows indicate TRPC1 bands.