Fig 2: Immunoblot detection of TREK/TRAAK family two-pore domain potassium channels in hippocampal tissue and hippocampal cultures.Western blots of TREK-1, TREK-2 and TRAAK from mouse hippocampus tissue (Hippo), HEK293 cells transfected with TREK-1 (HEK+TREK-1), TREK-2 (HEK+TREK-2), TRAAK (HEK+TRAAK) and hippocampal cultured cells (Cell Hippo). A, Upper panel, hippocampus (20 µg of protein) was probed with an anti-TREK-1 antibody with (+) or without (-) pre-absortion with the microsomal fraction of transfected HEK293 cells expressing TREK-2 and TRAAK, as described in Materials and Methods. HEK293 cells transfected with TREK-1 (10 µg of protein) and hippocampal cultured cells (20 µg of protein) were probed with the pre-absorbed anti-TREK-1. Lower panel, the pre-absorbed anti-TREK-1 was incubated with the corresponding antigenic peptide. B, Upper panel, hippocampus (20 µg of protein) was probed with (+) or without (-) pre-absortion with microsomal fraction of transfected HEK293 cells expressing TREK-1 and TRAAK, HEK293 cells transfected with TREK-2 (10 µg of protein) and hippocampal cultured cells (20 µg protein) were probed with the pre-absorbed anti-TREK-2. Lower panel, the pre-absorbed anti-TREK-2 was incubated with the corresponding antigenic peptide. C. Upper panel, hippocampus (20 µg of protein) was probed with an anti-TRAAK antibody with (+) or without(-) pre-absortion with microsomal fraction of transfected HEK293 cells expressing TREK-1 and TREK-2. HEK293 cells transfected with TRAAK (5 µg of protein) and hippocampal cultured cells (20 µg of protein) were probed with the pre-absorbed anti-TRAAK. Lower panel, the pre-absorbed anti-TRAAK was incubated with the antigenic peptide. D The levels of the major bands for each antibody in the hippocampus tissue and hippocampal cultured cells were calculated from the pixel intensity values (minus background) normalized to the pixel intensity values of HEK293- transfected cells and presented as optical density (OD). In this figure same batch of HEK293 transfected cells was used for hippocampus and cultured hippocampal cells, same batch of hippocampus tissue and hippocampal cultured cells were used to probe each antibody. Data correspond to one representative experiment of several experiments using different batches of samples with essentially the same results.

Fig 3: TREK-2 channels are preferentially expressed on IB4-positive small-sized maxillary TG neurons. (a, b) Fluorescent images show immunostaining for TREK-2 channels (a) and IB4-FITC staining (b) in the same maxillary region of a TG section. Scale bar: 50 µm. (c) Image in the box in A at expanded scale. (d) Image in the box in B at expanded scale. (e) Overlay image of (c) and (d). Scale bar: 20 µm. (f) and (g) Histogram of cell size distribution of TREK-2 immnoreactivity positive (f, n = 304) and IB4 positive (g, n = 466) maxillary TG neurons. (h) Percent of TREK-2-ir positive neurons (red bar, n = 11 TG sections) and IB4 positive neurons (green bar, n = 11 TG sectons) in total maxillary TG neurons. (i) Percent of neurons co-localized with TREK-2-ir and IB4 staining in all TREK-2-ir positive neurons (left bar, n = 11 TG sections) or in all IB4-positive neurons (right bar, n = 11 TG sections). Data represent Mean ± SEM.

Fig 4: Immunoreactivity of thermal K2P channels in lumbar dorsal root ganglia of rats. a Image shows TRAAK immunoreactivity (TRAAK-ir) in a L5 DRG section from an adult rat. b Overlay histograms show size distribution of TRAAK-ir positive (red bars, n = 890 cells) and total cells (open bars, n = 3178 cells). c Image shows TREK-1 immunoreactivity (TREK-1-ir) in a L5 DRG section from an adult rat. d Overlay histograms show size distribution of TREK-1-ir positive (red bars, n = 574 cells) and total cells (open bars, n = 2430 cells). e Image shows TREK-2 immunoreactivity (TREK-2-ir) in a L5 DRG section from an adult rat. f Overlay histograms show size distribution of TREK-2-ir positive (red bars, n = 630 cells) and total cells (open bars, n = 2928 cells). g Mean diameters of cells being positive to TRAAK-ir (1st bar, n = 890 positive cells), TREK-1-ir (2nd bar, n = 574 positive cells) and TREK-2-ir (3rd bar, n = 630 positive cells). h Percent of cells being positive of TRAAK-ir (1st bar, n = 8 sections), TREK-1-ir (2nd bar, n = 7 sections) and TREK-2-ir (3rd bar, n = 8 sections). Data in G and H represent Mean ± SEM, ***p < 0.001

Fig 5: a Mean mRNA levels of multiple K channels in conventionally grown Capan-1 cell cultures (light gray bars and symbols) and Capan-1 epithelial monolayers (dark gray bars and black symbols) (n = 4 for each condition). Aliases: KCNF1 (Kv5.1), KCNJ10 (Kir4.1), KCNJ2 (Kir2.1), KCNK1 (TWIK-1), KCNK10 (TREK-2), KCNK15 (TASK-5), KCNK16 (TALK-1), KCNK17 (TALK-2), KCNK2 (TREK-1), KCNK3 (TASK-1), KCNK5 (TASK-2), KCNK6 (TWIK-2), KCNK9 (TASK-3), KCNMA1 (KCa1.1), KCNN3 (KCa2.3), KCNN4 (KCa3.1), and KCNQ1 (Kv7.1). b RT-PCR analysis of KCNE1 mRNA in cell lysate from Capan-1 (Cap.) (H. refers to HPDE, another cell line). M. refers to the marker and Ctrl. to the control, with template substituted by H2O. c Western blots of Capan-1 protein samples showing expression of Kv7.1 (KCNQ1), TASK-2 (KCNK5), and TREK-2 (KCNK10). (Western blot for TREK-1 has been shown previously in Capan-1 cells [54].) *P < 0.05; **P < 0.01; ***P < 0.001