A Cantú syndrome mutation produces dual effects on KATP channels by disrupting ankyrin B regulation

Abstract

ATP-sensitive potassium (KATP) channels composed of Kir6.x and sulfonylurea receptor (SURs) subunits couple cellular metabolism to electrical activity. Cantú syndrome (CS) is a rare disease caused by mutations in the genes encoding Kir6.1 (KCNJ8) and SUR2A (ABCC9) that produce KATP channel hyperactivity due to a reduced channel block by physiological ATP concentrations. We functionally characterized the p.S1054Y SUR2A mutation identified in two CS carriers, who exhibited a mild phenotype although the mutation was predicted as highly pathogenic. We recorded macroscopic and single-channel currents in CHO and HEK-293 cells and measured the membrane expression of the channel subunits by biotinylation assays in HEK-293 cells. The mutation increased basal whole-cell current density and at the single-channel level, it augmented opening frequency, slope conductance, and open probability (Po), and promoted the appearance of multiple conductance levels. p.S1054Y also reduced Kir6.2 and SUR2A expression specifically at the membrane. Overexpression of ankyrin B (AnkB) prevented these gain- and loss-of-function effects, as well as the p.S1054Y-induced reduction of ATP inhibition of currents measured in inside-out macropatches. Yeast two-hybrid assays suggested that SUR2A WT and AnkB interact, while p.S1054Y interaction with AnkB is decreased. The p.E322K Kir6.2 mutation, which prevents AnkB binding to Kir6.2, produced similar biophysical alterations than p.S1054Y. Our results are the first demonstration of a CS mutation whose functional consequences involve the disruption of AnkB effects on KATP channels providing a novel mechanism by which CS mutations can reduce ATP block. Furthermore, they may help explain the mild phenotype associated with this mutation.

A rare gain of function HCN4 gene mutation is responsible for inappropriate sinus tachycardia in a Spanish family

“…CHO cells were grown in Ham-F12 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Caballero et al, 2010; Caballero et al, 2017; Pérez-Hernández et al, 2018; Alonso-Fernández-Gatta et al, 2021; Crespo-García et al, 2023). HEK-293 cells were cultured in Dulbecco’s modified Eagle’s (DMEM) medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin as previously described (Caballero et al, 2010; Crespo-García et al, 2023). The cultures were passed every 2–5 days using a brief trypsin treatment.…”

“…After settling to the bottom of the chamber, cells were perfused at ≈1 mL/min with external solution (see the composition below). Macroscopic currents were recorded at room temperature (21-23ºC) by means of the whole-cell patch-clamp technique using Axopatch-200B patch clamp amplifiers and pCLAMP software (Molecular Devices, San José, CA, USA) (Caballero et al, 2010; Caballero et al, 2017; Pérez-Hernández et al, 2018; Alonso-Fernández-Gatta et al, 2021; Nieto-Marín et al, 2022; Crespo-García et al, 2023) Recording pipettes were pulled from 1.0 mm o.d. borosilicate capillary tubes (GD1, Narishige Co., Ltd, Tokio, Japan) using a programmable patch micropipette puller (Model P-2000 Brown-Flaming, Sutter Instruments Co., Novato, CA, USA) and were heat-polished with a microforge (Model MF-83, Narishige).…”

“…Single channel currents were recorded in CHO cells at room temperature (21-23ºC) using the cell-attached patch-clamp configuration (Michels et al, 2005; Brandt et al, 2009; Caballero et al, 2010; Crespo-García et al, 2023). Using this configuration, the intracellular environment is completely preserved and channel activity can be measured in an intact cell.…”

“…The cultures were passed every 2-5 days using a brief trypsin treatment. For macroscopic current recordings, subconfluent cultures were transiently transfected with cDNA encoding WT or mutated HCN4 channels (1.6 µg) together with cDNA encoding the CD8 antigen (0.5 µg) by using X-tremeGENE™ HP (Roche Diagnostics, Rotkreuz, Switzerland) in CHO cells or Lipofectamine 2000 (Thermofisher Scientific, Waltham, MA, USA) in HEK-293 cells, with manufacturer instructions followed (Caballero et al, 2010;Caballero et al, 2017;Pérez-Hernández et al, 2018;Alonso-Fernández-Gatta et al, 2021;Crespo-García et al, 2023). In some experiments, WT and p.V240M HCN4 were cotransfected (WT+p.V240M) at a 0.5:0.5 ratio (0.8 µg each).…”

“…A biotinylation assay was conducted in CHO cells using procedures previously described (Crespo-García et al, 2023). At the point of 48 h after transfection of HCN4 WT or p.V240M, CHO cells were washed twice with ice cold phosphate-buffer saline (PBS) and biotinylated for 15 min at 4°C using PBS containing 0.5 mg/mL of EZ Link Sulfo-NHS-SS-Biotin (ThermoFisher Immunoresearch, West Grove, PA, USA).…”