Fig 2: The decrease of both TRPC4 and TRPC5 in the hippocampi of Cx3cr1CreER IL-10-/-mice. a RT-PCR results of TRPC isoforms in the hippocampus (n = 3 in each group, t-test). The marked significance is the comparison of the same isoform in the Cx3cr1CreER IL-10-/- with the littermate. b western blotting bands of TRPC1, TRPC3, TRPC4, and TRPC5 in littermates and in Cx3cr1CreER IL-10-/-mice. Bar graphs represent densitometric plots of protein expression in littermate and Cx3cr1CreER IL-10-/-mice in TRPC1 (c), TRPC3 (d), TRPC4 (e), and TRPC5 (f). Each dot represents a mouse. Bars represent mean ± SEM. n = 4 in each group, Significant differences were established by t-test. g Expression of TRPC1, TRPC3, TRPC4, and TRPC5 in the CA3 region of mouse hippocampal slices. Immunofluorescence images were captured with a 20 × objective. Green, immunoreactivity of TRPC1, TRPC3, TRPC4, and TRPC5; blue, nuclei stained with DAPI. Merged images of each TRPC isoform and DAPI staining. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 3: Overall structure of human TRPC5 (hTRPC5).(A, B) Inhibitory effect of clemizole (CMZ) (A) and HC-070 (B) on the extracellular calcium-induced intracellular calcium increase of cells with wild-type hTRPC5 over-expression measured by FLIPR calcium assay (data are shown as means ± standard error, n = 3 independent experiments). (C, D) Cryo-EM density maps of CMZ-bound hTRPC5 shown in side view (C) and top view (D). Subunits A, B, C, and D are colored in purple, light blue, orange, and gray, respectively. Lipids are colored in gold. The approximate boundary of the cell membrane is indicated by gray lines. TMD, transmembrane domain; ICD, intracellular cytosolic domain. (E) Ribbon diagram of a single subunit with secondary structure elements represented in different colors. ARD, ankyrin repeats domain; LHD, linker-helix domain; CH1, C-terminal helix 1; CH2, C-terminal helix 2. Figure 1—source data 1.Inhibition of WT hTRPC5 by clemizole or by HC-070.

Fig 4: Structure comparison between clemizole (CMZ)-bound human TRPC5 (hTRPC5), HC-070-bound hTRPC5, and apo hTRPC5 in transmembrane domain (TMD).(A) The overall conformational difference in TMD between these three structures. All the structures are shown as cartoon, with apo hTRPC5 colored in gray, CMZ-bound hTRPC5 in purple, and HC-070-bound hTRPC5 in light blue. (B) Close-up view of the structure variations of bottom of S3 segment and the S2-S3 linker between HC-070-bound and apo hTRPC5 structures.

Fig 5: Upregulation of TRPC5 in hippocampal excitatory neurons improving the learning and memory ability of Cx3cr1CreER IL-10-/-mice. a Schematic of experimental design and schedule. The animal experimental protocol indicated the time course of various interventions utilized during the experiment. b, c Schematics of AAV9-CaMKIIa-TRPC5 (b) and stereotaxic injection in the hippocampus (c). d The expression level of TRPC5 in hippocampus by Western blotting after AAV9 injection (n = 3). There was a significant increase in both escape latency (e) and average distance (f) in KO + TRPC5 compared to the KO group in the training stage. g Representative movement traces from the four groups on the test stage of the MWM task. The mice in the KO + TRPC5 group had more convergent paths in the test stage in the target quadrant, suggesting memory improvements. There was a significant increase in both the platform crossover times (h) and time spent in the target quadrant (i) in the KO + TRPC5 group in the test stage compared with the KO group. Each dot represents a mouse. Bars represent mean ± SEM. n = 5 in each group, Significant differences were established by two-way ANOVA (e, f) and one-way ANOVA (h, i). *P < 0.05, **P < 0.05