kcnj13 mouse rabbit alomone labs apc 125 wb  (Alomone Labs)

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: a–d Four views of the cryo-EM map of Kir7.1 obtained at 3.3 Å resolution, shown from the top ( d ) side ( a , b ) and bottom ( c ). The map is scaled to the atomic structure in ( e ) with each subunit color-coded. e Side view of the Kir7.1 structure model with two individual monomers colored green and purple (same orientation as ( b )). Only two chains of Kir7.1 model are shown for clarity. Structural features are labeled, including the outer, inner, pore and side helices, selectivity filter, extracellular region, G-loop, and the linker. Transmembrane (TMD) and cytoplasmic (CTD) domains are indicated. Dashed lines in the model indicate regions where model building was uncertain due to insufficient map resolution. f Sequence alignment of the selectivity filter region across all human Kir channels. A Met125 is pointed by a cyan triangle, and its conserved interacting residue Glu115 is labeled in yellow triangle. Selectivity filter is labeled as “SF” at the top. g Side view of Kir7.1 selectivity filter fitted into a cryo-EM map with potassium ions (purple spheres) visible inside the pore. Met125 and Glu115 are labeled. h Structural alignment of Kir7.1 (yellow) with homologous Kir channels (Kir2.2, blue; Kir3.2, salmon; Kir6.2, pink) highlights conserved architecture in the selectivity filter. A comparison of the Met125 in Kir7.1 versus Arg in other Kir channels is shown on the right. The distance between Glu and Arg in representative Kir2.2 (2.7 Å), is sufficient to form a salt bridge between these residues, however, the replacement of Arg with Met125 in Kir7.1 not only puts this residue 4.6 Å apart, but prevents a salt bridge formation due to a nature of thioether side chain.

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Cryo-EM Sample Prep, Labeling, Sequencing, Residue, Comparison

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: a Cryo-EM density of Kir7.1 in the extended (E-state) resolved at average resolution of 3.3 Å (grey). The transmembrane region is highlighted by a shaded background. b Cryo-EM density of Kir7.1 in the docked (D-state), resolved at average resolution of 3.9 Å (pink), shown at the same scale and view as E-state on the left. Total length of the complex in each state is labeled with its corresponding color. Extended map and docked map are aligned in cryoSPARC and their length were roughly estimated by using scale bar, showing extended map 115 Å and docked map 105 Å. Reference planes ( c – e ) correspond to panels below. c Structural alignment of the transmembrane domains between the E-state and D-state reveals a conserved selectivity filter but a curvature shift in the outer helix. d Superimposed cryo-EM density maps of the CTD in both states, viewed from the intracellular side, showing 45° clockwise rotation of the CTD in the D-state relative to the E-state. e Side view of the aligned Kir7.1 models highlighting the conformational rearrangement of the linker between CTD and TMD. The insert shows the structural transition of the linker region from a flexible loop (E-state) to an α-helix (D-state), suggesting its role in mediating domain movement.

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Cryo-EM Sample Prep, Labeling

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: a Side view of the cryo-EM density map of Kir7.1 in the D-state (purple), showing a prominent non-protein density corresponding to bound PIP₂ (blue). Inset: zoomed-in PIP₂ density. Maps and models are rotated by 45° for clarity. Endogenous PIP₂ is partially modeled where it fits the density. b Close-up of the PIP₂ binding pocket, highlighting positively charged residues within 3.5 Å of PIP₂ (Lys159, Arg162, Arg42, Lys164, Arg52, Arg54, and His26). PIP₂ is shown in blue and colored by atom (oxygen, red; nitrogen, dark blue; phosphorus, orange). c Sequence alignment of conserved PIP₂-binding motifs across human Kir channels, highlighting conserved residues coordinating phosphoinositide binding. Kir7.1 is shown at the top. Secondary structure elements (α-helices) are annotated, and conserved residues are boxed in red. Alignment was generated using Jalview and rendered with ESPript. d Quantification of Kir7.1 fold activation by progesterone (P4) in the presence or absence of PIP₂. Currents at −80 mV were normalized to baseline (control, no P4) recordings. No statistical differences (n.s.) were noted between conditions when progesterone was applied alone (P4+, PBP−), with PBP (P4+, PBP+), or consequently, immediately after PBP administration (P4+, PBP * +). Data are mean ± S.E.M., n = 3–7 cells. Post hoc Tukey’s tests were used for comparisons of mean values and statistical significance. e Representative whole-cell recordings from HEK293T cells expressing Kir7.1. First, application of the PIP₂ binding peptide (PBP, green) to sequester endogenous PIP₂ pool notably inhibited Kir7.1 responses. No synthetic diC8-PIP 2 was applied. Second, Kir7.1 was further stimulated with 10 μM progesterone (P4, plum) which led to a strongly increased Kir7.1 current, indicating that progesterone can activate the channel without PIP₂. Bath (black) indicates recordings in standard Krebs solution; control (blue) denotes recordings in KMeSO₃-based external solution. f Kir7.1 was first stimulated with 10 μM progesterone (P4, red), concurrently with 100 μM of synthetic intracellular diC8-PIP₂, producing an even stronger Kir7.1 activation, indicating a cooperativity between PIP₂ and progesterone. In both cases, ML418 (magenta), Kir7.1 inhibitor, was applied at the end of the recordings to return to the baseline.

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Cryo-EM Sample Prep, Binding Assay, Sequencing, Generated, Activation Assay, Control, Expressing

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: a Side view of the cryo-EM map of the Kir7.1 channel in the extended (E-state) conformation (light gray), with a PIP₂ molecule bound at the conserved interaction site (blue), and a distinct cholesterol (salmon) sits between two monomers. b Zoomed-in side view of E-state Kir7.1 (light gray) showing an additional non-protein density modeled as cholesterol (salmon). Maps and models are rotated by 10° for clearer visualization in ( b ) and ( c ). c Close-up view of the cholesterol-binding site between helices of adjacent subunits (left monomer-light gray; right monomer-dark gray). Cholesterol is shown with oxygen atoms in red and carbon in salmon. d Close-up view of the PIP₂ molecule (blue) bound in E-state (gray), colored by atom type: oxygen (red), nitrogen (blue), and phosphorus (orange). 45° rotation is performed for clearer visualization in ( d ) and ( e ). The structure of endogenous PIP₂ is partially modeled in the region to where it fits the density. e In PIP 2 binding pocket, positively charged residues (Arg42, Arg52, Arg54, Lys164, and Lys195) involved in PIP₂ coordination are highlighted. f Representative whole-cell recordings showing the effect of cholesterol (CHL) on P4-induced Kir7.1 activation. Top: 10 μM P4-induced activation is reduced by co-application of 100 μM CHL. Bottom: 30 μM P4 largely overcomes the inhibitory effect of 10 μM CHL. Chemical structures of progesterone (left) and cholesterol (right) are shown below. g Upper panel: representative Kir7.1 potassium currents (I K+ ) recorded from hKir7.1-expressing stable cell line (HEK293T-Kir7.1) in response to either P4 or P4 + CHL. Lower panel: Dose-response of P4 in the absence (black, n = 10) and the presence of 30 μM CHL (green, n = 9). EC₅₀ values were calculated from nonlinear regression fits. Data are presented as mean ± S.E.M. ( n = number of cells used per condition).

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Cryo-EM Sample Prep, Binding Assay, Activation Assay, Expressing, Stable Transfection

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: a Schematic illustration of the whole-cell patch-clamp configuration used for testing the indicated compounds under electrophysiological recording conditions. b Chemical structures of endogenous and synthetic steroids along with their stereoisomers, tested for Kir7.1 activation by whole-cell patch clamps in HEK293T cells. A progesterone molecule with carbon atom numbered, and steroid rings labeled is shown. c Dose-response curves of Kir7.1 activation by ENT-P4 and ENT-17OHPC. Left: 10 µM P4 is applied first, followed by 10 µM ENT-P4; Right: 10 µM ENT-17OHPC is applied. d Representative I K+ mediated by human Kir7.1 expressed in HEK293T cells in response to progesterone (P4), ENT-progesterone (ENT-P4), and ENT-17OHPC. ML418 was applied at the end of each recording to confirm current identity. EC₅₀ values were calculated from nonlinear regression fits. Data are presented as mean ± S.E.M. ( n = 9 cells per condition). e Fold increase in I K+ mediated by human Kir7.1 in response to the indicated compounds. N indicates the number of cells recorded per condition. Data are presented as mean ± S.E.M. Red boxes highlight the important double bond in steroid backbone essential for compound activity. Strong agonists are labeled in red. Blue boxes highlight a saturated A-ring, and the absent double bond in theinactive compounds labeled in blue. Compounds in black showed intermediate activity.

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Patch Clamp, Activation Assay, Labeling, Activity Assay

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: a Side view of the cryo-EM map of the Kir7.1 channel in the ENT-17OHPC-bound state (teal), with a diC8-PIP₂ molecule bound at the conserved interaction site (blue), and ENT-17OHPC (yellow) positioned between two monomers. b Zoomed-in side view of ENT−17OHPC-bound Kir7.1 (teal) showing an additional non-protein density modeled as ENT−17OHPC (yellow). Maps and models in ( b ) and ( c ) were rotated by 10° to improve visualization. c Close-up view of the ENT−17OHPC binding site between helices of adjacent subunits (left monomer, light teal; right monomer, dark teal). ENT-17OHPC is shown with oxygen atoms in red and carbon atoms in yellow. d Close-up view of the diC8-PIP₂ molecule (blue) bound in ENT-17OHPC-state (teal), colored by atom: oxygen (red), nitrogen (blue), and phosphorus (orange). Maps and models are rotated 45° for clearer viewing in ( d ) and ( e ). The diC8-PIP₂ model is partially modeled in the region to where it fits the density. e In diC8-PIP 2 binding pocket, positively charged residues (Arg42, Arg52, Arg54, Lys159, and Lys164) involved in PIP₂ coordination are highlighted. f Comparison of steroid binding sites between the extended Kir7.1 conformation and the ENT-17OHPC-bound Kir7.1 structure. Cholesterol is shown with oxygen atoms in red and carbon atoms in gray. ENT-17OHPC is colored with oxygen atoms in red and carbon atoms in cyan. g Surface hydrophobicity of the Kir7.1 cavity is shown, colored from hydrophobic (orange) to hydrophilic (teal). Left: in the ENT-17OHPC-bound Kir7.1 conformation, ENT-17OHPC (teal) occupies a predominantly hydrophobic pocket adjacent to the pore helices. Right: in the extended Kir7.1 conformation, cholesterol (gray) occupies a similar site that contacts both hydrophobic and polar residues. Dotted outlines indicate the approximate boundaries of each ligand-binding cavity. h Schematic of cholesterol (left, gray) and ENT-17OHPC (right, teal) illustrating their hydrophilic (teal box) and hydrophobic (orange box) regions. The oxygen-containing functional groups define the polar hydrophilic regions, whereas the hydrocarbon backbones represent the predominantly hydrophobic regions.

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Cryo-EM Sample Prep, Binding Assay, Comparison, Ligand Binding Assay, Functional Assay

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: a–c Comparative pore radius analysis of the a extended conformation, b docked conformation, and c ENT-17OHPC-bound conformation of Kir7.1, shown with their respective central pore profiles calculated by HOLE2 program. The pore is color-coded according to radius thresholds relative to water molecule size: red indicates a radius too narrow for water permeation, green represents a radius sufficient for a single water molecule, and blue corresponds to regions wide enough to accommodate multiple water molecules. The approximate membrane boundaries are shaded in gray. d Plot of pore radii along the ion conduction pathway for each conformation, indicating key structural changes in selectivity filter, central cavity, inner helix gate, and G-loop gate.

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Membrane

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: Extended state (gray), docked state (pink), ENT-17OHPC-bound state (teal), and a putative conductive state (blue) are shown to explain the mechanism of the Kir7.1 channel gating. Dashed lines indicate the curvature of the transmembrane helices across different channel states. The question marks in the putative conductive model denote the presence of a hypothetical high-potency agonist that may stabilize this conformation.

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques:

Journal: Nature Communications

Article Title: Bioactive lipid-mediated structural and functional regulation of the essential human potassium channel Kir7.1

doi: 10.1038/s41467-026-68819-0

Figure Lengend Snippet: Cryo-EM structures of representative Kir channels are shown with detailed views of three structural regions that contribute to inwardly rectification: the inner helix (pink), the G-loop gate (teal), and the CTD entrance (brown). Top: Kir7.1 (this study, PDB: 9PR5, gray). Middle: Kir3.2 (PDB: 6XIS; orange). Bottom: Kir2.1 (PDB: 7ZDZ; purple). V Valine (Val), I Isoleucine (Ile), D Aspartate (Asp), F Phenylalanine (Phe), M Methionine (Met), Y Tyrosine (Tyr), E Glutamate (Glu).

Article Snippet: Where indicated, the Kir7.1 antagonist ML418 (10 μM; Tocris, Inc cat no: 6889) and compounds were co-applied directly to the bath solution.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Genome-Wide CRISPR Screening Identifies Cellular Factors Controlling Nonviral Genome Editing Efficiency

doi: 10.1101/2025.03.12.642795

Figure Lengend Snippet: Base editing at the KCNJ13 W53X locus in a patient-derived Kir7.1 mutant RPE cells via lipid nanoparticles. (A) Schematic of base editing, performed by delivering ABE8e base editor to transduced Kir7.1 mutant RPE cells, knocked out for BET1L , MS4A13, and GJB2 . (B) Data is shown for three replicates (n=3) per treated condition ─ GJB2, MS4A13 , BET1L, untransduced mutant RPE (grey) and untreated/untransduced iPSC-RPE W53X/W53X (pink), shown as a correction (TAG->TGG), quantified as the % WT reads out of total reads (Y-axis) for different conditions. (C) Average Rb + fold change at −150 mV . (D) Schematic of Electrophysiology assay. (E-J) Kir7.1 expression (green) and its colocalization with a membrane marker, Na-K-ATPase (red), and a Current-Sweep time plot from a representative cell in different treatment solutions. (E) Untreated iPSC-RPE W53X/W53X (n=8). (F) Base edited iPSC-RPE W53X/W53X without KO of any gene (n=5). (G) Base edited MS4A13 -KO iPSC-RPE W53X/W53X (n=6). (H) Base edited GJB2 -KO iPSC-RPE W53X/W53X (n=4). (I) Base edited BET1L -KO iPSC-RPE W53X/W53X (n=4). (J) WT iPSC-RPE cells (n=15). Statistical significance was calculated using an ordinary one-way ANOVA Dunnett’s multiple comparisons test (A, B). *, p < 0.05; ****, p < 0.0001; ns, p > 0.05. RPE, retinal pigment epithelial cell; UTF, Untransfected; UT, Untransduced; WT, Wild-type; MT, Mutant; ICC, Immunocytochemistry; ANOVA, Analysis of Variance.

Article Snippet: Kir7.1 protein was detected using a mouse monoclonal primary antibody (Santa Cruz, sc-398810, 1:200).

Techniques: Derivative Assay, Mutagenesis, Expressing, Membrane, Marker, Immunocytochemistry