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

Abstract

The complex disorder Cantu syndrome (CS) arises from gain-of-function mutations in either or, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (K) channels, respectively. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determined the mechanism by which K function is altered by several substitutions in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/R1154W in TMD2. We engineered substitutions at their equivalent positions in rat SUR2A (D207E, Y981S, G985E, M1056I, and R1150Q/R1150W) and investigated functional consequences using macroscopic rubidium (Rb) efflux assays and patch-clamp electrophysiology. Our results indicate that D207E increases K channel activity by increasing intrinsic stability of the open state, whereas the cluster of Y981S/G985E/M1056I substitutions, as well as R1150Q/R1150W, augmented Mg-nucleotide activation. We also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. None of the D207E, Y981S, G985E, or M1056I substitutions had a significant effect on glibenclamide sensitivity. However, Gln and Trp substitution at Arg-1150 significantly decreased glibenclamide potency. In summary, these results provide additional confirmation that mutations in CS-associated SUR2 mutations result in K gain-of-function. They help link CS genotypes to phenotypes and shed light on the underlying molecular mechanisms, including consequences for inhibitory drug sensitivity, insights that may inform the development of therapeutic approaches to manage CS.

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

“…4 But the underlying mechanisms are mutation specifically and include enhanced Mg-ATP/ADP activation and increased intrinsic open probability. 7,29 Indeed, D207E, S1020P, S1054Y and R1154Q displayed gain-of-function characteristics in response to Mg-ATP. IC 50 values of R1154Q (0.64 ± 0.11 mmol/L and 0.75 ± 0.13 mmol/L for inward and outward current respectively) were in the same order as previously reported for the same SUR2A mutation (0.88 ± 0.19, 0.76 ± 0.12).…”

“…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). 29 sensitivity is blunted and now is in the micromolar range ( Figure 3).…”

Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders

“…At the moment, there is no treatment for CS available. However, the CS K ATP channel can be pharmaceutically targeted by several approved drugs, such as second-generation sulfonylureas, which are already used in clinical setups to inhibit GOF in the pancreatic K ATP isoforms involved in neonatal diabetes mellitus ( Ashcroft, 2010 ; McClenaghan et al, 2018 ). The successful validation of our CS KI zebrafish may provide the opportunity of phenotype-based screening to test the efficiency of these and further potential therapeutic compounds.…”

Implication of Potassium Channels in the Pathophysiology of Pulmonary Arterial Hypertension

“…To date, 12 different mutations in ABCC9 have been identified in Cantu Syndrome. Electrophysiological recordings have demonstrated that ABCC9 mutations are gain-of-function mutations [ 108 , 109 , 110 , 111 , 112 ]. The primary opening of SUR2A/Kir6.1 leads to systemic vasorelaxation and hypotension, and the secondary opening of SUR2A/Kir6.1 leads to compensatory cardiac hypertrophy and hypercontractility [ 113 ].…”