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Development of I_KATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant K_IR6.2 Based Channels for Treating DEND Syndrome.

ABSTRACT: Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the K_IR6.2 subunit of the I_KATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased I_KATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the K_IR6.2 WT and DEND mutant channels. Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, K_IR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships. Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective I_KIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC₅₀ values 27-37 μM). Levobetaxolol effectively inhibited WT and L164P (IC₅₀ values 22 μM) and Q52R (IC₅₀ 55 μM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC₅₀ 2-3 μM), while Q52R inhibition was 15-20% at 10 μM. Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated I_KATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve I_KATP inhibitor efficacy and specificity.

SUBMITTER: Houtman MJC

PROVIDER: S-EPMC8795863 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ATP-sensitive potassium (K(ATP)) channels play important roles in regulating insulin secretion, controlling vascular tone, and protecting cells against metabolic stresses. K(ATP) channels are heterooctamers of four pore-forming inwardly rectifying (Kir6.2) subunits and four sulfonylurea receptor (SUR) subunits. K(ATP) channels containing SUR1 (e.g. pancreatic) and SUR2A (e.g. cardiac) display distinct metabolic sensitivities and pharmacological profiles. The reported expression of both SUR1 and SUR2 together with Kir6.2 in some cells raises the possibility that heteromeric channels containing both SUR subtypes might exist. To test whether SUR1 can coassemble with SUR2A to form functional K(ATP) channels, we made tandem constructs by fusing SUR to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of K(ATP) channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2[N160D] (S1-ND), and SUR2A-Kir6.2[N160D] (S2-ND) in Xenopus oocytes, and studied the voltage dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Each tandem construct expressed alone supported macroscopic K(+) currents with pharmacological properties indistinguishable from those of the respective native channel types. Spermine sensitivity was low for S1-WT but high for S1-ND and S2-ND. Coexpression of S1-WT and S1-ND generated current components with intermediate spermine sensitivities indicating the presence of channel populations containing both types of Kir subunits at all possible stoichiometries. The relative abundances of these populations, determined by global fitting over a range of conditions, followed binomial statistics, suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities, suggesting that SUR1 and SUR2A randomly coassemble into functional K(ATP) channels. Further pharmacological characterization confirmed coassembly of not only S1-WT and S2-ND, but also of coexpressed free SUR1, SUR2A, and Kir6.2 into functional heteromeric channels.

Project description:BackgroundEstrogen has been shown to mediate protection in female hearts against ischemia-reperfusion (I-R) stress. Composed by a Kir6.2 pore and an SUR2 regulatory subunit, cardiac ATP-sensitive potassium channels (KATP) remain quiescent under normal physiological conditions but they are activated by stress stimuli to confer protection to the heart. It remains unclear whether KATP is a regulatory target of estrogen in the female-specific I-R signaling pathway. In this study, we aimed at delineating the molecular mechanism underlying estrogen modulation on KATP channel activity during I-R.Materials and methodsWe employed KATP knockout mice in which SUR2 is disrupted (SUR2KO) to characterize their I-R response using an in vivo occlusion model. To test the protective effects of estrogen, female mice were ovariectomized and implanted with 17?-estradiol (E2) or placebo pellets (0.1 ?g/g/day, 21-day release) before receiving an I-R treatment. Comparative proteomic analyses were performed to assess pathway-level alterations between KO-IR and WT-IR hearts.Results and discussionEchocardiographic results indicated that KO females were pre-disposed to cardiac dysfunction at baseline. The mutant mice were more susceptible to I-R stress by having bigger infarcts (46%) than WT controls (31%). The observation was confirmed using ovariectomized mice implanted with E2 or placebo. However, the estrogen-mediated protection was diminished in KO hearts. Expression studies showed that the SUR2 protein level, but not RNA level, was up-regulated in WT-IR mice relative to untreated controls possibly via PTMs. Our antibodies detected different glycosylated SUR2 receptor species after the PNGase F treatment, suggesting that SUR2 could be modified by N-glycosylation. We subsequently showed that E2 could further induce the formation of complex-glycosylated SUR2. Additional time-point experiments revealed that I-R hearts had increased levels of N-glycosylated SUR2; and DPM1, the first committed step enzyme in the N-glycosylation pathway. Comparative proteomic profiling identified 41 differentially altered protein hits between KO-IR and WT-IR mice encompassing those related to estrogen biosynthesis.ConclusionsOur findings suggest that KATP is likely a downstream regulatory target of estrogen and it is indispensable in female I-R signaling. Increasing SUR2 expression by N-glycosylation mediated by estrogen may be effective to enhance KATP channel subunit expression in I-R.

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Development of I_KATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant K_IR6.2 Based Channels for Treating DEND Syndrome.

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Development of IKATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant KIR6.2 Based Channels for Treating DEND Syndrome.

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Development of I_KATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant K_IR6.2 Based Channels for Treating DEND Syndrome.