Molecular dynamics simulations of KirBac1.1 mutants reveal global gating changes of Kir channels

Prokaryotic inwardly rectifying (KirBac) potassium channels are homologous to mammalian Kir channels. Their activity is controlled by dynamical conformational changes that regulate ion flow through a central pore. Understanding the dynamical rearrangements of Kir channels during gating requires high-resolution structure information from channels crystallized in different conformations and insight into the transition steps, which are difficult to access experimentally. In this study, we use MD simulations on wild type KirBac1.1 and an activatory mutant to investigate activation gating of KirBac channels. Full atomistic MD simulations revealed that introducing glutamate in position 143 causes significant widening at the helix bundle crossing gate, enabling water flux into the cavity. Further, global rearrangements including a twisting motion as well as local rearrangements at the subunit interface in the cytoplasmic domain were observed. These structural rearrangements are similar to recently reported KirBac3.1 crystal structures in closed and open conformation, suggesting that our simulations capture major conformational changes during KirBac1.1 opening. In addition, an important role of protein-lipid interactions during gating was observed. Slide-helix and C-linker interactions with lipids were strengthened during activation gating

Atomistic basis of opening and conduction in mammalian inward rectifier potassium (Kir2.2) channels

Potassium ion conduction through open potassium channels is essential to control of membrane potentials in all cells. To elucidate the open conformation and hence the mechanism of K+ ion conduction in the classic inward rectifier Kir2.2, we introduced a negative charge (G178D) at the crossing point of the inner helix bundle, the location of ligand-dependent gating. This forced open mutation generated channels that were active even in the complete absence of phosphatidylinositol-4,5-bisphosphate (PIP2), an otherwise essential ligand for Kir channel opening. Crystal structures were obtained at a resolution of 3.6 Å without PIP2 bound, or 2.8 Å in complex with PIP2. The latter revealed a slight widening at the helix bundle crossing (HBC) through backbone movement. MD simulations showed that subsequent spontaneous wetting of the pore through the HBC gate region allowed K+ ion movement across the HBC and conduction through the channel. Further simulations reveal atomistic details of the opening process and highlight the role of pore-lining acidic residues in K+ conduction through Kir2 channels

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

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 KIR6.2 subunit of the IKATP 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 IKATP, 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 KIR6.2 WT and DEND mutant channels. Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 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 IKIR6.2/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 IKATP 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 IKATP inhibitor efficacy and specificity

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression

Background: The inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) KIR2. 1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations. Methods: Molecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively. Results: PA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward IK1 at −50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM). Conclusions: 1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF

Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP(2)) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL(−)) with a distinct second site is required for high PIP(2) sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP(2) sensitivity, even in the absence of PL(−). Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP(2) (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL(−) binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP(2) site and explaining the positive allostery between PL(−) binding and PIP(2) sensitivity

Computational Identification of Novel Kir6 Channel Inhibitors

KATP channels consist of four Kir6.x pore–forming subunits and four regulatory sulfonylurea receptor (SUR) subunits. These channels couple the metabolic state of the cell to membrane excitability and play a key role in physiological processes such as insulin secretion in the pancreas, protection of cardiac muscle during ischemia and hypoxic vasodilation of arterial smooth muscle cells. Abnormal channel function resulting from inherited gain or loss-of-function mutations in either the Kir6.x and/or SUR subunits are associated with severe diseases such as neonatal diabetes, congenital hyperinsulinism, or Cantú syndrome (CS). CS is an ultra-rare genetic autosomal dominant disorder, caused by dominant gain-of-function mutations in SUR2A or Kir6.1 subunits. No specific pharmacotherapeutic treatment options are currently available for CS. Kir6 specific inhibitors could be beneficial for the development of novel drug therapies for CS, particular for mutations, which lack high affinity for sulfonylurea inhibitor glibenclamide. By applying a combination of computational methods including atomistic MD simulations, free energy calculations and pharmacophore modeling, we identified several novel Kir6.1 inhibitors, which might be possible candidates for drug repurposing. The in silico predictions were confirmed using inside/out patch-clamp analysis. Importantly, Cantú mutation C166S in Kir6.2 (equivalent to C176S in Kir6.1) and S1020P in SUR2A, retained high affinity toward the novel inhibitors. Summarizing, the inhibitors identified in this study might provide a starting point toward developing novel therapies for Cantú disease