DOI: 10.5061/dryad.7m0cfxq1g
title: Data from: KCNB1-Leptin receptor complexes couple electric and endocrine function in the melanocortin neurons of the hypothalamus

description: The neurons of the melanocortin system regulate feeding and energy
 homeostasis through a combination of electrical and endocrine mechanisms.
 However, the molecular basis for this functional heterogeneity is poorly
 understood. Here, a voltage-gated potassium (K+) channel named KCNB1
 (alias Kv2.1), formed stable complexes with the leptin receptor (LepR) in
 proopiomelanocortin (POMC) expressing neurons of the Arcuate nucleus of
 the hypothalamus (ARCPOMC). Mice lacking functional KCNB1 channels (NULL
 mice), exhibited enhanced activity of ARCPOMC neurons along with aberrant
 POMC production. In NULL neurons, canonical LepR-STAT3 signaling--which
 underlies POMC production--was impaired, whereas non-canonical insulin
 receptor substrate PI3K/Akt/FOXO1 and ERK signaling were significantly
 upregulated. Accordingly, the NULL animals were insensitive to anorexic
 stimuli induced by leptin administration and produced less adipose tissue
 and circulating leptin than WT animals. Taken together, these findings
 unveil an exquisite mechanism of metabolic regulation whereby synergistic
 control of neuronal excitability and endocrine function is achieved
 through the partnership between a K+ channel and a hormone receptor. As
 aberrant KCNB1 channels cause developmental and epileptic
 encephalopathies, these results may further identify a K+ channel as a
 causative link between epileptic and metabolic disorders.
description: Methods are properly described in the Methods section of the
 manuscript.
description: Original data used in the study entitled: "KCNB1-Leptin receptor
 complexes couple electric and endocrine function in the melanocortin
 neurons of the hypothalamus" by Elena Forzisi-Kathera-Ibarra, Chanmee
 Jo, Leonard Castillo, Christian Roser, Anika Gaur, Prachi Lad, Stefania
 Dutto, Ping-Yue Pan and Federico Sesti. Data available at:
 [https://doi.org/10.5061/dryad.7m0cfxq1g](https://doi.org/10.5061/dryad.7m0cfxq1g) All data used to generate the figures of the study, are stored in this repository except: 1) transcriptome data (Figure 2), which are stored in the GEO metadata repository of the National Institutes of Health and 2) original images used for the figures of the paper which are subject to copyright. For each figure of the paper, the corresponding original data are contained in a folder named after the figure's number, i.e. FIGURE 1 etc.. Each FIGURE folder is organized in sub-folders, sub-sub-folders etc. corresponding to a specific experimental condition. Please note: 1. The genotype "NULL" is often called "KO". 2. The genotype "WT" can also be called NTG 3. "KI" refers to a genotype not presented in the manuscript and should therefore be ignored. A) Western blots are named with the date of experiment, the primary antibody (i.e. KCNB1 or LepR or c-fos etc.) and the genotype of each lane of the blot moving from left to right. They can contain additional information such as for example the name of who carried out the experiment. For example a blot named "Elena 7-7-22 WTKOKI" means that Elena (Forzisi) made the experiment on 7-7-22. The genotypes from left to right are WT, NULL and KI. B) Confocal images are either in canonical formats (JPEG, PNG etc.) or in Nikon ND2 format. They can be imported in ImageJ or read with other, freely available software. C) Nomarski images are in .jpg or .png format. Images named with a progressive number followed by [number]-[X] followed by the excitation light channel used for the immunofluorescence. The "[number]-[X]" is added by the software and can be ignored. The initial number indicates images of the same area excited/visulaized with different lights, such as GFP, CY3 etc.: 4[1]-[X]-DAPI.jpg = Image 4, DAPI visualization 4[0]-[X]-GFP.jpg = Image 4, green channel 4[2]-[X]-CY3.jpg = Image 4, far-red channel Therefore, the composite image is indicated with the number alone, thus 4.jpg in the example. A different set of images would therefore be named as: 5[1]-[X]-DAPI.jpg 5[0]-[X]-GFP.jpg 5[2]-[X]-CY3.jpg etc. D) ELISA data are organized in Excel files generated by the Tecan Infinite M Nano+ plate reader. These files contain the parameters of the instrument (wavelenght, bandwidth etc.) and calibration data with associated graph. The experimental data are segregated by the date of the experiment and named after the genotype (WT, KO etc.) and the experimental condition (control, leptin etc.). They may contain associated graphs. E) Data used to construct the graphs of the figures of the associated manuscript are in prism.format.   The files contain the data named after the genotype/experimental condition and the associated graph  with the statistical analysis. FIGURE FOLDERS: FIGURE 1_FINAL_New_102824 contains three sub-folders. Sub-folder "kcnb1 1wt3 with ab 111022_confocal" contains confocal images of WT hypothalami immunostained for KCNB1. Individual images are named as kcnb1hypo 111022.nd2, kcnb1hypo 111023.nd2 etc. Sub-folder "kcnb1 WT free floating 061323" contains two sub-sub-folders. Sub-sub-folder named "confocal images", contains images of hypothalamic slices stained for KCNB1 and POMC. Images are named KCNB1_POMC_WT (number).nd2. The sub-sub-folder named "free floating final 050423" contains Nomarski images of hypothalamic slices stained for KCNB1, contained in the sub-sub-sub-folder "Kcnb1" or images of slices stained for KCNB1 and POMC, contained in the sub-sub-sub-folder "pomc Kcnb1". Images from brain 12 and brain 13 are contained in the corresponding sub-sub-sub-sub-folders. Individual images are named as described in C). Similar considerations apply to sub-folder kcnb1 Null free floating 061323. FIGURE 3_S2_FINAL_New_102824 contains the sub-folder named "mice 20 weeks" with images of WT (in sub-sub-folder WT) or NULL (in sub-sub-folder NULL) of WT or Null mice. Single images are progressively numbered. The sub-folder "MRI" contains the data for MRI in an Excel file. GraphPad prism files named "ELISA_Bloodglucose_Fat_analysis" that contains the data of blood glucose and fat measurements and "weight_Food_Leptinexperiments_GTT" contain the data of weight, food intake leptin concentration and glucose tollerance tests. FIGURE 4_FINAL_New_102824 contains the Prism file weight_Food_Leptinexperiments_GTT that contains the results of leptin-sensitivity experiments including the associated graphs and statistical analysis. FIGURE 5_FINAL_New_102824 contains 4 sub-folders. The sub-folder "leptin control wt_KO_2623_exp2" and "Leptin_control wt_KO\_ exp1" contain four sub-sub-folders for each experiment. They are named "ko pomc control", "ko pomc leptin" "wt pomc control" and "wt pomc leptin". They indicate immunofluorescent experiments in hypothalamic NULL (ko) or WT slices stained with POMC antibody in the absence/presence of leptin. Each sub-sub-folder contains sub-sub-sub-folders named "brain X" with X a number, that contain individual Nomarski images obtained from the indicated brain. Files are named as described in C). The sub-folder named "Leptin_control wt_KO\_ exp1" contains data segregated for genotype in the two sub-sub-folders, "ko" and "WT". Each contains two sub-sub-folders named "pomc_stat3_control" and "pomc_stat3_control" that contain immunofluorescent experiments in hypothalamic NULL (ko) or WT slices stained for POMC or pSTAT3 in the absence/presence of leptin. The Nomarski files are organized in "brainX" sub-sub-sub-sub-folders as described above. Files are named as described in C). The sub-folder named "pomc WB" contains original Westerns taken from lysates of 4 weeks old or 24 weeks old hypothalami, that are contained in the corresponding "4th week" and "24th week" sub-sub-folders. Westerns are named as described in A) For example the blot named "pomc kokoleptin 071323_1(Chemiluminescence).jpg" in the sub-sub-folder "24th week", refers to an experiment carried out on 13 July 2023, of hypothalamic lysates stained with pomc antibody. From left to right: NULL (ko) control and NULL (ko) leptin. FIGURE 6_FINAL_New_102824 contains five sub-folders. Sub-folders "Erk WB", "FOXO WB", "leptinR WB" and "STAT3 WB" contain Western blots that are named as described in A), with the date of experiment, the primary antibody (i.e. STAT3, FOXO1 etc.) and the genotype of each lane of the blot moving from left to right. Additional conditions may be indicated such as for example the name of the experimenter or the absence/presence of leptin. Sub-folder "alpha MSH elisa" contains ELISA measurements of alpha MSH in two Excel files. The file ELISA AKT_PAKT.xlsx contains ELISA measurements of Akt and pAkt. The Excel files contain the parameters of the plate reader, calibration curves and experiments segregated by date, genotype and experimental condition. FIGURE 7_FINAL_New_102824 contains a sub-folder named "RNA SCOPE LEPR_KCNB1_POMC DATA" which contains a sub-sub-folder named "overlap photo analysis" with representative images showed in Figure 7 for LepR_KCNB1_POMC overlap. Figure 7 contains 2 lif files "RNA scope images" and "wt3tile scan" which represents all the confocal images taken with Leica TCS SP8 tauSTED 3X for RNA scope experiments of WT and Null brains showed in Figure 7. There is an Excel file named "RNA scope data analysis" and the graphpad prims file "RNA scope analysis" which contains the data used for the analysis, graphical representations and statistical analyses. FIGURE 8_FINAL_New_102824 contains confocal and Nomarski images of 3 immunofluorescent experiments. Sub-foldes "EXP1" and "EXP2" contain confocal images. The names of the single files reflect the antibodies used for the staining (KCNB1 and POMC) and are progressively numbered. Example: KCNB1_POMC (1).nd2. The sub-folder "KCNB1 WT POMC free floating_EXP3" contains Nomarski images organized in 3 sub-sub-folders. Sub-sub-folders "brain12" and "brain 13" contain individual images named as described in C). Sub-sub-folder "additional images" contains additional images whose files are named as described in C). FIGURE 9_S10_FINAL_New_102824. This folder contains the original data used to construct FIGURE 9 and its associated supplementary figure, FIGURE S10. The data are organized in three sub-folders. Sub-folder "Neurons_EXP1" contains immunofluorescence images from experiment 1. Sub-sub-folder "Confocal KCNB1 LepR Map2" contains composite confocal images of primary neurons stained for KCNB1, LepR and Map2. Files are named "LEPR_KCNB1_MAP2 (X).nd2" with X a progressive number. The sub-sub-folder "Nomarski KCNB1 LepR Map2" contains confocal images of primary neurons stained for KCNB1 and LepR. Files are named "LEPR_KCNB1_MAP2 (X).nd2" with X a progressive number. The sub-folder "Neurons_EXP2" contains composite confocal files organized in the sub-sub-folder named "neurons 022023 letpin kcnb1 map2" that are named as described. The sub-folder "leptinR_KCNB1_COIP" contains Western blot data of the co-IPs of the LepR with KCNB1, integrin-beta5 and integrin-beta1 and co-IPs of KCNB1 with the LepR, integrin-beta5 and integrin-beta1. Experiments are organized in sub-sub-folders named with the date of the experiment, i.e. "COIP date". Individual files are named following the usual convention described in A). FIGURE 10_S9_FINAL_New_102824 is organized in 2 sub-folders "Figure 10" and "Figure S9". Figure 10 contained the sub-sub-folder named "cfos WB" which contains the Western Blot membranes named as described in A), the Excel analysis file "WB analysis collective" and the graphpad prism file "WB_IF_analysis_comprehensive" representing Western Blot experiments shown in figure 10 of WT and Null animals with and without leptin treatments. Figure 10 contains another sub-sub-sub-folder named "cFos" which contains all the confocal images taken with Leica TCS SP8 tauSTED 3X for RNASCOPE experiments of WT and Null with Leptin or not shown in figure 10. Figure 10 folder also contains the graphpad prism file named "Analysis c-Fos RNA scope". The file contains original data segregated by date the experiment, genotype and experimental condition along with associated graphs and statistical analyses. Figure S9 sub-folder is essentially organized as Figure 8. It contains three sub-sub-folders of immunofluorescence data and one sub-sub-folder of Western blot data. The sub-sub-folder "cfos WB" contains Western blots stained for c-FOS. Westerns are named as described in A). The sub-sub-folders "leptin control wt_KO_2623_exp2", "Leptin_control wt_KO\_ exp1" and "Leptin_control_KO_WT_free floating 062322_exp3" contain Nomarski images segregated for genotype (as done for the sub-folder "Leptin_control wt_KO\_ exp1" described in Figure 5). Files in "brainX" sub-sub-sub--folders are further contained in "10X" and "20X" sub-sub-sub-sub-folders corresponding to the magnification of the optic of the microscope. The  file "WB Analysis collective.prism" contains the densitometric analysis of the Westerns, segregated by date the experiment, genotype and experimental condition along with associated graphs and statistical analyses. FIGURE S1_FINAL_New_102824 contains Western blots of wt or Null hypothalamic lysates stained for KCNB1 antibody and lysates stained for LepR antibody. The files contained in the sub-folder "kcnb1 WB"  are named as described in A), with the date of experiment, the primary antibody (i.e. STAT3, FOXO1 etc.) and the genotype of each lane of the blot moving from left to right. Additional conditions may be indicated such as for example the name of the experimenter or the absence/presence of leptin. The files contained in the sub-folder “LepR antibody validation WB” contain the western blots of the validation of the antibody as shown in the figure. FIGURE S6_FINAL_New_102824 contains images extracted from the BRAIN ATLAS of the Allen Institute used for the FIGURE S3 and for the analysis of overlap done with ImageJ. The folder contains the Excel file "AllenInstitute_singlecellsqedata" which contains the data of cell types characterization directly from the Allen Institute and the data obtained during the overlap analysis done with ImageJ. The folder also contains the graphpad prism file "Analysis singlecell Allen institute" which hold the data and the graph obtained for the figure. FIGURE S7_FINAL_New_102824 contains immunofluorescence and Western blot data organized in the sub-folders "AGRP staining" (immunofluorescence) and "ARPG WB" (Western blots). The Western files are named as described in A). The immunofluorescence files are also essentially organized as described in Figure 5. The sub-sub-folder "5c6 kcnb1 argp 800 500 030722" contained in "AGRP staining" contains Nomarski images of hypothalamic slices stained for KCNB1 and AgRP that are organized in three "brain X" sub-sub-sub-folders. Files are named as described in C). The sub-sub-folder "030322" contains confocal and Nomarski images contained in the sub-sub-sub-folders "wt agrp kcnb1 031122_confocal" and "4 WT 12 KCNB1 AGRP 800(kcnb1), 500 (agRP)". All images are immunofluorescence stainings with KCNB1 and AgRP antibody. Images are organized in "brain X" sub-sub-sub-sub-folders as described. The sub-sub-folder "5c6 kcnb1 argp 800 500 030722" contains Nomarski images of hypothalamic slices stained with KCNB1 and AgRP antibody that are organized in "brain X" sub-sub-sub-sub-folders as described. FIGURE S3_FINAL_New_102824 contains two sub-folders. Sub-folder named "Leptin_control_WT_Null_free floating 2623_EXP1" contains immunofluorescence images of stainings for pSTAT3 or POMC for WT or NULL in the absence/presence of leptin for experiment 1. Data are segregated for genotype in the sub-folders "WT" or "NULL". In turn these sub-folders contain two subfolders, one named "genotype pomc_stat3 control" that contains data taken in the absence of leptin and one "genotype pomc_stat3 leptin" that contains data taken in the presence of leptin. Nomarski images are contained in sub-sub-sub-folders named "brainX" (where X is the number of the brain). The individual files are named as described in C). For example the file 1[0]-[X]-GFP.jpg contained in Leptin_control_WT_Null_free floating 2623_EXP1/WT/wt pomc_stat_leptin/Brain4 is a Nomarski image immunofluorescent for GFP (corresponding to pSTAT3) of WT brain #4 taken in the presence of leptin. The same data classification applies to sub-folder Leptin_control_WT_Null_free floating 062322_EXP2 (corresponding to experiment 2). FIGURE S5_FINAL_New_102824 contains immunofluorescence data of alpha-MSH staining. The folder is organized in two sub-folders: "092623 alpha MSH 200 500" and "100423 alpha MSH 200 500". Sub-folder "092623 alpha MSH 200 500" contains two sub-sub-folders, named WT and KO (corresponding to NULL) of Nomarski images of hypothalamic slices stained with alpha-MSH antibody that are named following the usual criteria as described in C). The sub-folder "100423 alpha MSH 200 500" contains "free-floating" images. These are organized in two sub-sub-folders named "aMSH 10x" and "aMSH 20x" corresponding to the 10x or 20x magnification. Each of these sub-sub-folders contains data segregated in "WT" or "KO" sub-sub-sub-folders that contain Nomarski images named "brainX.jpg" with X the number of the brain. Some images may contain the extra suffix "up" or "down" which refers to the side of the slice. FIGURE S9_FINAL_New_102824 Contains immunofluorescence co-localizations of KCNB1 and Leptin receptor in hypothalamic slices of WT. The data are confocal images contained in the sub-folder named "kcnb1 lepR 080423_staining". Confocal files are named progressively example: 1012.nd2. Cropped images are converted in .png format and are named "LepR_KCNB1" followed by the magnification of the optic and the channel of the confocal. Example: the file "LepR_KCNB1_40x_FITC.png" is a slice stained with KCNB1 and LepR antibodies, taken at 40x using the green channel (FITC).