Cantu syndrome-associated SUR2 (ABCC9) mutations in distinct structural domains result in K_ATP channel gain-of-function by differential mechanisms

Abstract

The complex cardiovascular disorder Cantu Syndrome arises from gain-of-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (K ATP ) channels. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determine the mechanism by which K ATP function is altered by several mutations in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/W in TMD2. Mutations were engineered at their equivalent position in rat SUR2A (D207E, Y981S, G985E, M1056I and R1150Q/W) and functional effects were investigated using macroscopic rubidium ( Mg-nucleotide activation. The response of mutant channels to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS, was also tested. There was no major effect on glibenclamide sensitivity for the D207E, Y981S, G985E or M1056I mutations, but glutamine and tryptophan substitution at R1150 resulted in significant decreases in potency.

Glibenclamide treatment in a Cantú syndrome patient with a pathogenic ABCC9 gain‐of‐function variant: Initial experience

“…At the cellular level, there is good evidence that sulfonylureas act more potently on SUR1 channels than on SUR2, and that many CS mutations result in further decrease in SUR2 sensitivity to these drugs (Gribble & Ashcroft, ; McClenaghan et al, ). Thus glibenclamide or other available sulfonylureas are probably not ideal drugs for CS, and actions on SUR1 in the pancreas could yield significant side effects.…”

“…Mutations were introduced into a rat SUR2A (pCMV_rSUR2A; GenBank accession No. D83598.1) cDNA construct using site‐directed mutagenesis (McClenaghan et al, ). Cultured Cosm6 cells, transfected with wild‐type pcDNA3.1_mKir6.2 (0.6 μg; GenBank accession No.…”

“…Cultured Cosm6 cells, transfected with wild‐type pcDNA3.1_mKir6.2 (0.6 μg; GenBank accession No. D50581.1) and wild‐type or mutant pCMV_rSUR2A constructs (1 μg) were analyzed by patch clamp (McClenaghan et al, ). Dose–response curves (Figure S1A,B) confirmed that, while the intrinsic sensitivity to inhibition by ATP (in the absence of Mg 2+ ) was no different from wild‐type channels, R1116H mutant channels were more strongly activated by MgATP and MgADP, explaining the overall gain‐of‐function.…”

Glibenclamide and HMR1098 normalize Cantú syndrome‐associated gain‐of‐function currents

“…In the absence of nucleotides, D207E sensitivity for glibenclamide was in the nanomolar range (42.8 ± 11.4 nmol/L) and similar as for wild‐type channels (29.5 ± 11.1 nmol/L) . However, in the presence of nucleotides (0.15 mmol/L Mg‐ATP and 0.5 mmol/L Mg‐ATP) and thus better mimicking the physiological conditions, glibenclamide sensitivity is blunted and now is in the micromolar range (Figure ).…”

Three‐dimensional facial morphology in Cantú syndrome

“…Cantú syndrome (CS; OMIM #239850) is a rare autosomal dominant condition characterized by a wide constellation of clinical features, namely coarse facial features, congenital hypertrichosis, osteochondrodysplasia and extensive cardiovascular anomalies including cardiomegaly, patent ductus arteriosus (PDA), pericardial effusion and dilated and torturous cerebral blood vessels (Grange et al, ; Grange, Lorch, Cole, & Singh, ; Leon Guerrero et al, ; Scurr et al, ). CS is caused by gain‐of‐function (GoF) pathogenic variants in ABCC9 and, less commonly, in KCNJ8 , which encode the regulatory (SUR2) and pore‐forming (Kir6.1) subunits, respectively, of ATP‐sensitive potassium (K ATP ) channels(Brownstein et al, ; Cooper et al, ; Harakalova et al, ; McClenaghan et al, ; van Bon et al, ). Notably, a majority of cases are considered to harbor de novo variants.…”