PIP2 Binding Research Articles

The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.

Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer

Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca2+, ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP2) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (Po) shows little voltage dependence and is stable over several minutes. Po is high without addition of exogenous PIP2, but is largely blunted by treatment with poly-l-Lysine, a polycation that masks PIP2 headgroups. These results indicate that PIP2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP2 binding affinity of nvTRPM2 under physiological conditions.

Crosstalk between regulatory elements in disordered TRPV4 N-terminus modulates lipid-dependent channel activity

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and the network of antagonistic regulatory elements it encodes. These modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.

The membrane electric field regulates the PIP2-binding site to gate the KCNQ1 channel

Voltage-dependent ion channels underlie the propagation of action potentials and other forms of electrical activity in cells. In these proteins, voltage sensor domains (VSDs) regulate opening and closing of the pore through the displacement of their positive-charged S4 helix in response to the membrane voltage. The movement of S4 at hyperpolarizing membrane voltages in some channels is thought to directly clamp the pore shut through the S4-S5 linker helix. The KCNQ1 channel (also known as Kv7.1), which is important for heart rhythm, is regulated not only by membrane voltage but also by the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). KCNQ1 requires PIP2 to open and to couple the movement of S4 in the VSD to the pore. To understand the mechanism of this voltage regulation, we use cryogenic electron microscopy to visualize the movement of S4 in the human KCNQ1 channel in lipid membrane vesicles with a voltage difference across the membrane, i.e., an applied electric field in the membrane. Hyperpolarizing voltages displace S4 in such a manner as to sterically occlude the PIP2-binding site. Thus, in KCNQ1, the voltage sensor acts primarily as a regulator of PIP2 binding. The voltage sensors' influence on the channel's gate is indirect through the reaction sequence: voltage sensor movement → alter PIP2 ligand affinity → alter pore opening.

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.

New drug-like small molecule antagonizes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in patients with conotruncal heart defects

ObjectivesConotruncal heart defects (CTDs) are highly heritable, and approximately one-third of all congenital heart defects are due to CTDs. Through post-analysis of GWAS data relevant to CTDs, a new putative signal transduction pathway, called Vars2-Pic3ca-Akt, associated with CTD has been hypothesized. Here, we aimed to validate the Vars2-Pic3ca-Akt pathway experimentally by measuring Vars2 and PIP3 in patients with CTDs and controls, and to construct a PIP3 inhibitor, as one of harmful-relevant CTD pathogenesis, through an Akt-based drug design strategy. Methodsrs2517582 genotype and relative Vars2 expression in 207 individuals were determined by DNA sequencing and qPCR respectively, and free plasma PIP3 in 190 individuals was quantified through ELISA. An Akt-pharmacophore feature model was used to discover PIP3 antagonists with multiple computational and drug-like estimation tools. ResultsCTD pathogenesis due to Vars2-Pic3ca-Akt overstimulation was confirmed by elevated Vars2 and PIP3 in patients with CTDs. We identified a new small molecule, 322PESB, that antagonizes PIP3 binding. This molecule was prioritized via virtual screening of 21 hypothetical small molecules and it showed minimal RMSD change, high binding affinity andlower dissociation constant than PIP3-Akt complex by 1.99 Kcal/Mol, thus resulting in an equilibrium shift toward 322PESB-Akt complex formation. Moreover, 322PESB exhibited acceptable pharmacokinetics and drug likeness features according to ADME and Lipinski's rule of five classifiers. This compound is the first potential drug-like molecule reported for patients with CTDs with elevated PIP3. ConclusionPIP3 is a useful diagnostic biomarker for patients with CTDs. The Akt-pharmacophore feature model is a feasible approach for discovery of PIP3 signalling antagonists. Further 322PESB development and testing are recommended.

PO-01-206 MOLECULAR MODELING OF CLINICAL KCNJ2 MUTATIONS REVEALS STRUCTURE-FUNCTION IMPACT ON PIP2 BINDING SITE

Binding phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for Kir2.1(encoded by KCNJ2), function. Disease associated KCNJ2 mutations, such as those associated with Andersen-Tawil Syndrome (ATS), may exert molecular dysfunction by interrupting PIP2 binding, however a full-length molecular model of Kir2.1 has not been developed to explore this hypothesis. Elucidate the molecular structure of Kir2.1 and determine the functional impact of clinical KCNJ2 mutations on PIP2 binding using experimental and improved molecular dynamic (MD) method. Whole-cell patch clamp experiments of WT-Kir2.1 and selected mutations were performed on stably transfected HEK293 cells using a standard protocol. A full-length Kir2.1 tetramer model (open and closed conformational states) based on Kir2.2 cryo-EM structure using MOE was created. Mutations (R67Q, G300D, R218L) were individually introduced to the channel, and a mutagenesis analysis was performed. Initially, MD simulation for 100ns was performed for wild-type (WT) and mutated structures (GROMACS software). We expanded the model to include +/- bound PIP2. Due to > 77% degree of homology of Kir2.2 (6m84) with Kir2.1, we developed a MD protocol with Kir2.2 as test system. The 2.5% free PIP2 molecules were released into the membrane and MD simulation was performed for 700ns using Amber software. Homomeric WT demonstrated normal Kir2.1 function while mutations G300D, R67Q, and R218L showed complete loss of function. Mutational analysis revealed that residue changes result in disruption of hydrogen bonding and affects the structural conformation at the residue sight and caused fluctuation of residues in other regions, i.e., selectivity filter and pore domain. The improved MD protocol validated Kir2.2 PIP2 binding sites. Moreover, we found mutation disruption of hydrogen bonds and hydrophobic interaction analysis between the channel and PIP2 molecules revealed significant structural changes. We developed the first full length MD model of Kir2.1 and found that ATS KCNJ2 mutations disrupt normal conformation and hydrogen bonding. Further, we validated PIP2 binding sites which illustrate the robustness of our method, and this technique can be applied to delineate Kir2.1 molecular PIP2 binding sites. Combined with experimental validation, our modeling will increase our understanding of structure-function relationships of KCNJ2 clinical mutations.

Isotherm kinetics of PIP2 bound gelsolin inactivation

Actin monomers (G-actin) and filaments (F-actin) have dynamical rearrangement thus manage cellular motility, division and transport processes. The gelsolin (GSN) regulates the remodeling of cytoskeleton. After the activation of GSN by calcium ions, it can sever actin filaments then capped at its barbed end. In the cytoplasm, GSN manages the cellular motions and morphology. Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in signal transduction and the regulation of the actin cytoskeleton by regulation of actin-binding proteins. GSN can bind to PIP2 and thus can be localized in the near of the plasma membrane and released from the end of F-actin. We test here with isoperibol calorimetry the enthalpy change, within the interplay between GSN and F-actin under nano-, micro- and millimolar calcium concentrations and express the importance of PIP2 binding for the inactivation of GSN. As we have demonstrated here that PIP2 binding stabilizes the structure of gelsolin and reduces its actin monomer binding activity under nanomolar calcium as the typical cytoplasmic calcium concentration of resting cells. The gelsolin shows partial activity under micromolar and total activity with strong responses under millimolar calcium. If gelsolin-capped filaments point at the plasma membrane helps the binding between gelsolin and PIP2, and hence, filament uncapping in case of resting cells. We presume that the low free calcium concentration keeps on the structure of gelsolin which is able to bind actin within the cooperativity of actin bound calcium. Gelsolin can help to manage monomer pool far from the membrane and it can be linked to a basic sensory mechanism which drives the direction of filament growth in the near of the membrane.

N-WASP is competent for downstream signaling before full release from autoinhibition.

Allosteric regulation of intrinsically disordered proteins (IDPs) is still vastly understudied compared to the counterpart of structured proteins. Here, we used molecular dynamics simulations to characterize the regulation of the IDP N-WASP by the binding of its basic region with inter- and intramolecular ligands (PIP2 and an acidic motif, respectively). The intramolecular interactions keep N-WASP in an autoinhibited state; PIP2 binding frees the acidic motif for interacting with Arp2/3 and thereby initiating actin polymerization. We show that PIP2 and the acidic motif compete in binding with the basic region. However, even when PIP2 is present at 30% in the membrane, the acidic motif is free of contact with the basic region ("open" state) in only 8.5% of the population. The very C-terminal three residues of the A motif are crucial for Arp2/3 binding; conformations where only the A tail is free are present at a much higher population than the open state (40- to 6-fold, depending on the PIP2 level). Thus, N-WASP is competent for Arp2/3 binding before it is fully freed from autoinhibition.

Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association

Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention.

Why Does Synergistic Activation of WASP, but Not N-WASP, by Cdc42 and PIP2 Require Cdc42 Prenylation?

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

The second PI(3,5)P2 binding site in the S0 helix of KCNQ1 stabilizes PIP2-at the primary PI1 site with potential consequences on intermediate-to-open state transition.

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

PIP2 binding at the voltage sensor domain facilitates KCNQ1 VSD activation and gating.

The cardiac KCNQ1 channel is involved in cell hyperpolarisation, and loss-of-function mutations are associated with Long QT syndrome (LQTS), which can cause sudden cardiac arrest. The KCNQ1 channel is gated by the changes in transmembrane voltage and crucially requires the binding of two phosphatidylinositol-4,5-bisphosphate (PIP2) molecules to open the channel. The recent structure of KCNQ1 has revealed a PIP2 binding site at the voltage-sensor domain (VSD). In addition, previous modelling and electrophysiological studies have proposed another PIP2 binding site at the pore domain. Using enhanced sampling molecular dynamics simulation, we show that PIP2 binding at the VSD facilitates its activation, whereas PIP2 interaction of the pore facilitates VSD-pore coupling by straightening the S6 helix. Our simulation proposes that PIP2 binding at the VSD acts as a counter charge pausing point during the voltage-dependent activation process, lowering the energy barrier during the S4 translocation. Together, our work highlights the importance of PIP2 at the VSD as an activator to the VSD activation, in addition to our previous knowledge as the key coupler between the VSD and the pore.

TRP434 and TRP435 residues are crucial for sensing calcium ions and PI(4,5)P2 molecules in TRPC5 channel.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. Among 7 subunits, TRPC4, 5 channels are well known to be potentiated by direct bindings of calcium ion and phosphatidylinositol 4,5-biphosphate. Both calcium and PIP2 were therefore also implicated in activation and desensitization of the channel. The binding site for calcium ion was revealed but PIP2 binding site remains controversial. Previously, we found two tryptophan residues in TRPC4 channel as a candidate by which calcium sensing in S2-S3 linker is delivered to channel gating. Here, we suggest the WW site in TRPC5 channel conducts delivering of calcium sensing like in TRPC4, and also acts as a potential PIP2 binding site. We made two single mutants (W434A, W435A) and a double mutant (WW/AA) from human TRPC5 channel. Wild-type and mutant TRPC5 channels were expressed in HEK293 cells for macroscopic current recordings in whole-cell or inside-out mode. Generally, our data demonstrate W434 and W435 residues participate in PIP2 binding and calcium sensing. Interestingly, when co-expressed with human muscarinic acetylcholine receptor type 3 (mAChR3) and stimulated by carbachol, W434A channel didn’t show desensitization characteristically observed in wild-type TRPC5 channel. Loss of desensitization in W434A channel implies that it has lost the ability of sensing PIP2 hydrolysis by PLC or the ability of phosphorylation by PKC. The latter possibility is ruled out because the C-terminus of the channels we used were truncated so they do not contain the phosphorylation site. Recently, PIP2-bound ion channel structures were revealed including TRPs. Many channels had PIP2 molecules near S3, S4, and S5 helices. Our electrophysiological data also indicate that PIP2 binds near S3 helix in TRPC5 channel, and the WW site is a specific candidate for sensing PIP2 and calcium.

Untangling the inseparable life of an ABC transporter and a potassium channel: Towards understanding the structure-function relationship in the KATP channel complex.

KATP channels are unique protein complexes of a potassium channel, Kir6.1 or Kir6.2, and an ABC transporter, SUR1, SUR2A or SUR2B (splice variants of SUR2), in a 4:4 stoichiometry. Broadly expressed in many cell types and tissues, KATP channels couple changes in cellular metabolic activity with membrane excitability to control a wide range of physiological processes, including hormone secretion, control of vascular tone, learning and memory, and cardiac and neuronal protection against ischemic insults. In pancreatic β-cells KATP channels formed by Kir6.2 and SUR1 control insulin secretion, and gain- or loss-of-function mutations in these channels cause neonatal diabetes or congenital hyperinsulinism, respectively. Physiological activity of pancreatic KATP channels is regulated by intracellular ATP and ADP. Non-hydrolytic binding of ATP to Kir6.2 inhibits channel activity, while MgATP and MgADP binding to the nucleotide binding domains (NBDs) of SUR1 stimulates channel activity. Besides ATP and ADP, membrane phospholipids in particular PIP2 interacts with Kir6.2 and stimulates channel activity. In addition, pharmacological inhibitors and activators act on SUR1 to modulate channel activity. Although Kir6.2 and SUR1 harbor the primary binding sites for specific physiological and pharmacological ligands, both subunits participate in ligand regulation. How do the potassium channel and the silent ABC transporter work together to decipher signals from different ligands for gating has long been a major question in the KATP channel field. Here, I present structural and functional evidence that SUR1 contributes directly to ATP and PIP2 binding via its TMD0 and L0 linker to enhance Kir6.2 sensitivity to both ligands. Moreover, SUR1 regulates Kir6.2 activity allosterically via its dynamic interaction with the N-terminus of Kir6.2.

The bisphosphonate zoledronic acid is a TRPV1 channel inhibitor.

The heat and capsaicin sensor, the transient receptor potential vanilloid type 1 (TRPV1) channel, is critically involved in pain sensation. Using compounds that activate TRPV1, such as capsaicin (CAP), produces analgesic effects. Hence the channel is a promising target for the management of pain conditions. However, when TRPV1 channels are sensitized in neuropathic pain conditions, inhibitors could be a better option for pain management. We have obtained evidence supporting that the bisphosphonate compound zoledronic acid (ZOL) acts as an inhibitor of the TRPV1 channel. ZOL is typically used in the treatment of bone diseases reducing bone mass loss, deterioration in bone microarchitecture and fracture risk, having as a collateral effect a decrease in pain sensation. We have observed, expressing TRPV1 in Xenopus laevis oocytes followed by patch-clamp recordings in inside-out configuration, that ZOL at doses ranging from 1 nM up to 30 μM reduces channel conductance and inhibits the activity of the channel deeply. Only high doses of CAP (≥ 10 μM) allow for recover of TRPV1 currents after ZOL treatment. Molecular modeling followed by docking analysis suggests that ZOL could potentially bind to TRPV1 at the vanilloid pocket and the PI(4,5)P2 binding site. Furthermore, like capsazepine, ZOL (100 nM) is able to eliminate the depression observed by applying CAP (1 μM) on synaptic transmission in the dentate gyrus. In addition, when ZOL is use during the induction of synaptic plasticity mediated by TRPV1, it eliminates plasticity. The compound did not affect the endocannabinoid production machinery suggesting that the ZOL is acting specifically on TRPV1 to hinder plasticity. Together, our evidence strongly supports that ZOL can inhibit TRPV1 activity.

Full activation of WASP, not N-WASP, by Cdc42 and PIP2 requires Cdc42 prenylation.

WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GPTase Cdc42, to relieve auto-inhibition before they can stimulate the initiation of actin polymerization. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the upstream WH1 domain. The basic region also participates in Cd42 binding, especially for WASP; consequently, Cdc42 binding significantly compromises the ability of WASP, but not N-WASP, to bind PIP2. PIP2 binding of WASP is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the full activation of WASP and N-WASP may contribute to their different function roles.

Structural-functional characterization of KCNJ2 variants associated with ventricular arrhythmic syndrome phenotypes.

KCNJ2 encodes inwardly rectifying potassium channels (Kir2.1) and plays a key role in establishing resting membrane potential and cell excitability. Mutations in KCNJ2 have been linked to several clinical phenotypes associated with ventricular arrhythmia and sudden death including Andersen-Tawil syndrome (ATS) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)-like phenotypes. It is unclear if the underlying structural-functional relationship is the cause for different clinical phenotypes. Here we combine computational molecular modeling and ion current analysis of four patients’ KCNJ2 mutations from the Inherited Arrhythmia Clinic: i) 2 (R218L, G300D) with ATS phenotype, ii) and 2 (R67Q, V227F) who present with a CPVT-like phenotype. Whole-cell patch clamp experiments of WT-Kir2.1 and four selected mutations were performed on stably transfected HEK293 cells using a standard protocol. For structure-based investigation, a full-length 3D Kir2.1 tetramer model was developed and mutations were individually introduced to the channel. The molecular dynamics (MD) simulation was carried out for 50 ns to assess each mutation's impact on overall channel conformation compared to WT. Electrophysiology experiments of homomeric CPVT-like R67Q-Kir2.1 and V227F-Kir2.1 displayed no current showing that these are loss-of-function mutations. Similarly, homomeric ATS mutations G300D-Kir2.1 and R218L-Kir2.1 also showed loss of function and no measurable current. The MD simulations revealed that WT conformation of Kir2.1 was more stable compared to mutant structures. The conformational changes may disrupt PIP2 binding with the channel that subsequently alter the pore region and block the ion conductance path. Combined ionic current and molecular dynamic analysis provided deeper structure-function and mechanistic insight into the molecular mechanisms of disease. Comparative conformational analysis of WT and mutant revealed the channel's overall behavior and impact on channel functioning. This will further help in understanding mutation-specific channel pharmacology and developing targeted therapeutic strategies.

A novel KIR-specific KATP channel opener.

KATP channels have diverse roles, including regulation of insulin secretion, protection against cardiac stress responses, and blood flow regulation, and are therefore excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the specific assemblies of pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against various KATP channel subclasses. We generated 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures in both the open and closed states and identified a potential agonist binding pocket in a functionally critical area of the channel in the vicinity of a palmitoylated Kir6.2-C166 residue and the PIP2 binding region. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds were performed. The top 15 ‘hits’ were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes and of them (70862985) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 respectively 5 µM and 1 µM). Remarkably, at 15-100 µM, compound 70862985 had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1 or Kir3.1/Kir3.4 channels and at 100 µM minimally affected Na+ currents in TE671 medulloblastoma cells. We examined whether 70862985 can correct Kir6.2 mutants with a decreased open probability, which is typically associated with congenital hyperinsulinism. Each of the Kir6.2-F55L, Kir6.2-T62M and Kir6.2-R301C mutants were associated with reduced Tl+ fluxes from Kir6.2/SUR1 transfected HEK-293 cells and were relatively insensitive to diazoxide. Kir6.2-R301C, by contrast, was strongly activated by 70862985. 70862985 is a Kir6.2 opener that does not affect the structurally similar Kir6.1 channel. This, to our knowledge, is the first Kir-specific channel opener.

Structural insights into the complex between Arf1 and ASAP1 at the membrane surface by NMR.

ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of GTP bound to the small GTP-binding protein Arf1. Simultaneous binding of the Pleckstrin homology (PH) domain of the ArfGAP ASAP1 to membranes containing the phosphatidylinositol phosphate PIP(4,5)P2 and to Arf1.GTP is key for maximum GTP hydrolysis, but is not fully understood. Recently, we showed that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain triggers an allosteric conformational switch (PIP switch) involving regions distant from the membrane interface. Here, we examined the interface between Arf1 and ASAP1 PH at the membrane surface of nanodiscs by Nuclear Magnetic Resonance (NMR) methods. Chemical Shift Perturbations (CSPs) observed upon complex formation and distance restraints obtained from Paramagnetic Relaxation Enhancement (PRE) NMR experiments between multiple MTSL-tagged Arf1 mutants and isotopically labeled ASAP1 PH were combined to build a model of the Arf1:ASAP1 PH complex at the membrane. Interestingly, the PIP switch resides at the interface between Arf1 and ASAP1 PH, shedding light on how PIP binding to ASAP1 PH may affect GTP hydrolysis.