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GITR is a TNF receptor, and its activation promotes immune responses and drives antitumor activity. The receptor is activated by the GITR ligand (GITRL), which is believed to cluster receptors into a high-order array. Immunotherapeutic agonist antibodies also activate the receptor, but their mechanisms are not well characterized. We solved the structure of full-length mouse GITR bound to Fabs from the antibody DTA-1. The receptor is a dimer, and each subunit binds one Fab in an orientation suggesting that the antibody clusters receptors. Binding experiments with purified proteins show that DTA-1 IgG and GITRL both drive extensive clustering of GITR. Functional data reveal that DTA-1 and the anti-human GITR antibody TRX518 activate GITR in their IgG forms but not as Fabs. Thus, the divalent character of the IgG agonists confers an ability to mimic GITRL and cluster and activate GITR. These findings will inform the clinical development of this class of antibodies for immuno-oncology.

Membrane protein function is regulated by the lipid bilayer composition. In many cases the changes in function correlate with changes in the lipid intrinsic curvature (c0), and c0 is considered a determinant of protein function. Yet, water-soluble amphiphiles that cause either negative or positive changes in curvature have similar effects on membrane protein function, showing that changes in lipid bilayer properties other than c0 are important—and may be dominant. To further investigate the mechanisms underlying the bilayer regulation of protein function, we examined how maneuvers that alter phospholipid head groups effective “size”—and thereby c0—alter gramicidin (gA) channel function. Using dioleoylphospholipids and planar bilayers, we varied the head groups’ physical volume and the electrostatic repulsion among head groups (and thus their effective size). When 1,2-dioleyol-sn-glycero-3-phosphocholine (DOPC), was replaced by 1,2-dioleyol-sn-glycero-3-phosphoethanolamine (DOPE) with a smaller head group (causing a more negative c0), the channel lifetime (τ) is decreased. When the pH of the solution bathing a 1,2-dioleyol-sn-glycero-3-phosphoserine (DOPS) bilayer is decreased from 7 to 3 (causing decreased head group repulsion and a more negative c0), τ is decreased. When some DOPS head groups are replaced by zwitterionic head groups, τ is similarly decreased. These effects do not depend on the sign of the change in surface charge. In DOPE:DOPC (3:1) bilayers, pH changes from 5→9 to 5→0 (both increasing head group electrostatic repulsion, thereby causing a less negative c0) both increase τ. Nor do the effects depend on the use of planar, hydrocarbon-containing bilayers, as similar changes were observed in hydrocarbon-free lipid vesicles. Altering the interactions among phospholipid head groups may alter also other bilayer properties such as thickness or elastic moduli. Such changes could be excluded using capacitance measurements and single channel measurements on gA channels of different lengths. We conclude that changes gA channel function caused by changes in head group effective size can be predicted from the expected changes in c0.

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.

SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel expressed in cardiac myocytes and human gastrointestinal (GI) smooth muscle cells (SMCs). NaV1.5 contributes to electrical excitability in the heart and slow waves in the gut. NaV1.5 is also mechanosensitive, and mechanical force modulates several modes of NaV1.5’s voltage-dependent function. NaV1.5 mutations in patients with cardiac arrhythmias and gastrointestinal diseases lead to abnormal mechano- and voltage-sensitivity. Membrane permeable amphipathic drugs that target NaV1.5 in the heart and GI tract alter NaV1.5 mechanosensitivity (MS), suggesting that amphipaths may be a viable therapeutic option for modulating NaV1.5 function. We therefore searched for membrane-permeable amphipathic agents that would modulate NaV1.5 MS with minimal effect on NaV1.5 voltage-gating intact to more selectively target mechanosensitivity. We used two methods to assess NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macropatch and whole-cell Na+ current parameters with and without force. The pressure- and shear-mediated peak current increase and acceleration were effectively abolished by capsaicin. Capsaicin abolished the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of MS makes capsaicin is a novel modulator of NaV1.5 MS and a promising therapeutic candidate.

© 2019 Biophysical Society Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

Ghovanloo et al. combined in silico and in vitro approaches to determine the mechanism by which CBD inhibits the skeletal muscle Na+ channel Nav1.4. Their findings suggest that CBD acts directly, by binding inside the Nav1.4 pore, as well as indirectly, by modulating membrane elasticity., Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.

Cannabis sativa contains active constituents called phytocannabinoids. Some phytocannabinoids are psychotropic and others are not. The primary non-psychotropic phytocannabinoid is cannabidiol (CBD), which is proposed to be therapeutic against many conditions, including muscle spasms. Mechanisms have been proposed for the action of CBD on different systems, involving multiple targets, including the voltage-gated sodium channel (Nav) family, which are heavily implicated in many of the conditions CBD has been reported to relieve. In this study, we investigated the modulatory mechanism of CBD on Nav1.4. Based on previous results, we tested the hypothesis that CBD mechanism of action involves: 1) modulation of membrane elasticity, which indirectly contributes to Nav inhibition; and 2) physical block of the Nav pore. We first performed molecular dynamic (MD) simulations to visualize CBD effects and localization inside the membrane, and then performed NMR to verify the MD results, showing CBD localizes below membrane headgroups. Then, we performed a gramicidin-based fluorescence (GFA) assay that showed CBD alters membrane elasticity. Next, we used site-directed mutagenesis in (F1586A) and around (WWWW) the Nav1.4 pore. Removing the local anesthetic binding site with F1586A reduced CBD block of INa. Occluding the fenestrations with WWWW blocked CBD access from the membrane into the Nav1.4 pore. However, stabilization of inactivation, via CBD-induced changes in membrane elasticity persisted, in WWWW. To investigate the potential therapeutic value of CBD against some Nav1.4 channelopathies, we used a pathogenic variant of Nav1.4, P1158S, known to cause myotonia and periodic paralysis. We found CBD reduces excitability in both wild-type and the mixed myotonia/periodic paralysis variant. Our in-vitro/in-silico results suggest that CBD may have therapeutic value against myotonia. Because Nav1.4 is crucial to skeletal muscle contraction, we used rat diaphragm myography and found the presence of saturating levels of CBD reduces skeletal muscle contraction.SUMMARYWe used multidisciplinary approaches to show the mechanism and pathway by which CBD inhibits the skeletal muscle, Nav1.4. Our results suggest CBD modulates membrane elasticity and directly interacts with Nav1.4 within its pore.

Made available in DSpace on 2020-12-12T01:23:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Optical Society Universidade Estadual Paulista Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) National Institutes of Health Resveratrol (RVT) derivatives (10a-i) were designed, synthesized, and evaluated for their potential as gamma-globin inducers in treating Sickle Cell Disease (SCD) symptoms. All compounds were able to release NO at different levels ranging from 0 to 26.3%, while RVT did not demonstrate this effect. In vivo, the antinociceptive effect was characterized using an acetic acid-induced abdominal contortion model. All compounds exhibited different levels of protection, ranging from 5.9 to 37.3%; the compound 10a was the most potent among the series. At concentrations between 3.13 and 12.5 µM, the derivative 10a resulted in a reduction of 41.1–64.3% in the TNF-α levels in the supernatants of macrophages that were previously LPS-stimulated. This inhibitory effect was higher than that of RVT used as the control. In addition, the compound 10a and RVT induced double the production of the gamma-globin chains (γG + γA), compared to the vehicle, using CD34+ cells. Compound 10a also did not induce membrane perturbation and it was not mutagenic in the in vivo assay. Thus, compound 10a emerged as a new prototype of the gamma-globin-inducer group with additional analgesic and anti-inflammatory activities and proving to be a useful alternative to treat SCD symptoms. São Paulo State University (UNESP) School of Pharmaceutical Sciences University of Campinas (UNICAMP) Hematology and Hemotherapy Center Weill Cornell Medical College Department of Physiology and Biophysics São Paulo State University (UNESP) School of Pharmaceutical Sciences FAPESP: 2012/50359-2 FAPESP: 2014/00984-3 CNPq: 304731/2017-0 National Institutes of Health: GM21342

Drugs do not act solely by canonical ligand-receptor binding interactions. Amphiphilic drugs partition into membranes, thereby perturbing bulk lipid bilayer properties and possibly altering the function of membrane proteins. Distinguishing membrane perturbation from more direct protein-ligand interactions is an ongoing challenge in chemical biology. Herein, we present one strategy for doing so, using dimeric 6-bromo-2-mercaptotryptamine (BrMT) and synthetic analogues. BrMT is a chemically unstable marine snail toxin that has unique effects on voltage-gated K+ channel proteins, making it an attractive medicinal chemistry lead. BrMT is amphiphilic and perturbs lipid bilayers, raising the question of whether its action against K+ channels is merely a manifestation of membrane perturbation. To determine whether medicinal chemistry approaches to improve BrMT might be viable, we synthesized BrMT and 11 analogues and determined their activities in parallel assays measuring K+ channel activity and lipid bilayer properties. Structure-activity relationships were determined for modulation of the Kv1.4 channel, bilayer partitioning, and bilayer perturbation. Neither membrane partitioning nor bilayer perturbation correlates with K+ channel modulation. We conclude that BrMT's membrane interactions are not critical for its inhibition of Kv1.4 activation. Further, we found that alkyl or ether linkages can replace the chemically labile disulfide bond in the BrMT pharmacophore, and we identified additional regions of the scaffold that are amenable to chemical modification. Our work demonstrates a strategy for determining if drugs act by specific interactions or bilayer-dependent mechanisms, and chemically stable modulators of Kv1 channels are reported.

Quantitative investigations into functional properties of purified ion channel proteins using standard electrophysiological methods are challenging, in particular for the determination of average ion channel behavior following rapid changes in experimental conditions (e.g., ligand concentration). Here, we describe a method for determining the functional activity of liposome-reconstituted K+ channels using a stopped-flow fluorometric ion flux assay. Channel activity is quantified by measuring the rate of fluorescence decrease of a liposome-encapsulated fluorophore, specifically quenched by thallium ions entering the liposomes via open channels. This method is well suited for studying the lipid bilayer dependence of channel activity, the activation and desensitization kinetics of ligand-dependent K+ channels, and channel modulation by channel agonists, blockers, or other antagonists.

Clinically important antiarrhythmic drugs may act by modifying primarily physical properties of the cell membrane., Amiodarone is a widely prescribed antiarrhythmic drug used to treat the most prevalent type of arrhythmia, atrial fibrillation (AF). At therapeutic concentrations, amiodarone alters the function of many diverse membrane proteins, which results in complex therapeutic and toxicity profiles. Other antiarrhythmics, such as dronedarone, similarly alter the function of multiple membrane proteins, suggesting that a multipronged mechanism may be beneficial for treating AF, but raising questions about how these antiarrhythmics regulate a diverse range of membrane proteins at similar concentrations. One possible mechanism is that these molecules regulate membrane protein function by altering the common environment provided by the host lipid bilayer. We took advantage of the gramicidin (gA) channels’ sensitivity to changes in bilayer properties to determine whether commonly used antiarrhythmics—amiodarone, dronedarone, propranolol, and pindolol, whose pharmacological modes of action range from multi-target to specific—perturb lipid bilayer properties at therapeutic concentrations. Using a gA-based fluorescence assay, we found that amiodarone and dronedarone are potent bilayer modifiers at therapeutic concentrations; propranolol alters bilayer properties only at supratherapeutic concentration, and pindolol has little effect. Using single-channel electrophysiology, we found that amiodarone and dronedarone, but not propranolol or pindolol, increase bilayer elasticity. The overlap between therapeutic and bilayer-altering concentrations, which is observed also using plasma membrane–like lipid mixtures, underscores the need to explore the role of the bilayer in therapeutic as well as toxic effects of antiarrhythmic agents.

The endoplasmic reticulum (ER) forms a branched, dynamic membrane tubule network that is vital for cellular function. Branching arises from membrane fusion facilitated by the GTPase atlastin (ATL). Many metazoan genomes encode for three ATL isoforms that appear to fulfill partially redundant function despite differences in their intrinsic GTPase activity and localization within the ER; however, the underlying mechanistic differences between the isoforms are poorly understood. Here, we identify discrete temporal steps in the catalytic cycle for the two most dissimilar isoforms, ATL1 and ATL3, revealing an overall conserved progression of molecular events from nucleotide binding and hydrolysis to ATL dimerization and phosphate release. A crystal structure of ATL3 suggests a mechanism for the displacement of the catalytic Mg2+ ion following guanosine triphosphate (GTP) hydrolysis. Together, the data extend the mechanistic framework for how GTP hydrolysis drives conformational changes in ATL and how the cycle is reset for subsequent rounds of catalysis.

Background: The mechanism of ligand gating in physiologically important cyclic nucleotide-modulated channels is unknown. Results: We constructed and purified a chimeric ion channel with activity modulated by cAMP and used it to measure ligand-binding energetics. Conclusion: cAMP binds with high lipid-dependent affinity to the chimeric channel. Significance: The availability of a good protein preparation enables assays that shed light on ligand gating.Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.

Poly-unsaturated fatty acids (PUFAs) alter the function of many membrane proteins, whereas monounsatured fatty acids generally are inert. We previously showed that docosahexaenoic acid (DHA) at pH 7 decreases the bilayer stiffness, consistent with an amphiphile-induced increase in elasticity, but not with a negative change in curvature; oleic acid (OA) was inert (Bruno, Koeppe and Andersen, Proc. Natl. Acad. Sci. USA 104:9638–9643, 2007). To further explore how PUFAs and other amphiphiles may alter lipid bilayer properties, and thus membrane protein function, we examined how changes in acyl chain unsaturation, and head group charge and size, alter bilayer properties, as sensed by bilayer-spanning gramicidin A (gA) channels of different lengths. Compared to DHA, the neutral DHA-methyl ester has reduced effects on bilayer properties, and 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine (PDPC) forms bilayers that are softer than dioleoylphosphatidylcholine (DOPC). The changes in channel function are larger for the short gA channels, indicating that changes in elasticity dominate over changes in curvature. We altered the fatty acid protonation by titration: docosahexaenoic acid (DHA) is more potent at pH 9 (relative to pH 7) and is inert at pH 4; OA, which was inert at pH 7, becomes a potent modifier of bilayer properties at pH 9. At both pH 7 and 9, DHA and OA produced larger changes in the lifetimes of the short gA channels, demonstrating that they increase lipid bilayer elasticity when deprotonated—though OA promotes the formation of inverted hexagonal phases at pH 7. The positively charged oleylamine (OAm), which has a small head-group and therefore should be a negative curvature promoter, inhibited gA channel function, with similar reductions in the lifetimes of the short and long gA channels, indicating a curvature-dominated effect. Monitoring the single-channel conductance, we find that the negatively charged fatty acids increase the conductance by increasing the local negative charge around the channel, whereas the positively charged OAm has no effect. These results suggest that deprotonated fatty acids increase bilayer elasticity by reversibly adsorbing at the bilayer/solution interface.

The marine snail toxin BrMT, a 6-bromo-2-mercaptotryptamine dimer, is a powerful, hydrophobic modulator of Kv1 and Kv4 voltage-gated potassium channels. It remains unclear how BrMT modulates these diverse channels, and given BrMT's hydrophobicity, whether it alters potassium channel function by perturbing lipid bilayer properties. We synthesized BrMT and 11 analogs to determine structure-activity relationships, and create more chemically stable ion channel modulators. We found the biological activity was retained when the labile disulfide bond between BrMT's indole groups was replaced with an ether or alkane linkage.

The coupling between membrane proteins and their surrounding lipid bilayer make understanding the membrane lipid regulation of membrane proteins key to understanding their function. The membrane lipid regulation of membrane protein function ranges from specific lipid binding to more general lipid bilayer regulation. The latter mechanism arises from energetic coupling between the lipid bilayer and membrane protein where protein conformational change will alter the organization of the adjacent bilayer. To gain insight into, and tease apart, the specific and general mechanism(s) of membrane protein regulation by the membrane lipids, we examine how the prototypical bacterial potassium channel, KcsA, is regulated by bilayer modifying compounds and drugs using a fluorescence-based technique. The concentration ranges at which the compounds alter the lipid bilayer is determined using gramicidin channels as probes. Comparing the concentration ranges at which molecules alter KcsA function and lipid bilayer properties, enable us to determine whether the underlying mechanism primarily involves specific interactions with KcsA or is bilayer-mediated. Most compounds tested slowed activation and accelerated inactivation at concentrations below those where they alter bilayer properties, suggesting that KcsA likely has a non-specific site that allows hydrophobic molecules to bind and alter channel properties. This site may be the fenestrations proposed to be located on the cytosolic side of aqueous pore of potassium channels (and evident in the x-ray structure). The same compounds alter KcsA function at the concentrations where they alter lipid bilayer properties. These, bilayer-modifying, molecules slow recovery from inactivation at concentrations at which they alter bilayer properties. These results suggest that conformational changes responsible for recovery from inactivation are regulated by a bilayer-mediated mechanism. Our results point to a complicated relationship between direct and bilayer-mediated mechanisms, which both are rather non-specific and may affect a diverse range of ion channels.

The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator–activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer–water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs’ partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer–dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and underscore the importance of exploring bilayer effects of candidate drugs early in drug development.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2) or PIP(2)] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP(2) and other phosphoinositides. Here, we report a novel requirement for PIP(2) in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP(2) levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP(2) levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP(2) and PIP(2)-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP(2). Neutralization of positively charged amino acids abolished EGFR/PIP(2) interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR auto-phosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP(2) and point to the basic JD's critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP(2) may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.

Phosphorylation of the Kir3 channel by cAMP-dependent protein kinase (PKA) potentiates activity and strengthens channel-PIP(2) interactions, whereas phosphorylation by protein kinase C (PKC) exerts the opposite effects (Keselman et al., Channels 1:113-123, 2007; Lopes et al., Channels 1:124-134, 2007). Unequivocal identification of phosphorylated residues in ion channel proteins has been difficult, but recent advances in mass spectrometry techniques have allowed precise identification of phosphorylation sites (Park et al., Science 313:976-979, 2006). In this study, we utilized mass spectrometry to identify phosphorylation sites within the Kir3.1 channel subunit. We focused on the Kir3.1 C-terminal cytosolic domain that has been reported to be regulated by several modulators. In vitro phosphorylation by PKA exhibited a convincing signal upon treatment with a phosphoprotein stain. The phosphorylated C terminus was subjected to mass spectrometric analysis using matrix-assisted lased desorption/ionization-time of flight mass spectroscopy (MS). Peptides whose mass underwent a shift corresponding to addition of a phosphate group were then subjected to tandem MS (MS/MS) in order to confirm the modification and determine its precise location. Using this approach, we identified S385 as an in vitro phosphorylation site. Mutation of this residue to alanine resulted in a reduced sensitivity of Kir3.1* currents to H89 and Forskolin, confirming an in vivo role for this novel site of the Kir3.1 channel subunit in its regulation by PKA.

Many neurotransmitters activate G-protein-gated inwardly rectifying K+ (Kir3) channels by stimulating G-protein-coupled receptors. However, in native systems, only receptors coupled to pertussis-toxin (PTX)-sensitive G proteins (Gi/Go) have been shown to be able to activate Kir3 channels through the βγ subunits of G proteins (Gβγ), whereas activation of receptors coupled to PTX-insensitive G proteins such as Gq or Gs do not activate Kir3 channels. The question remains as to how signaling specificity is achieved and what are its key determinants. In this study, we have used the Xenopus oocyte expression system to investigate specific activation of Kir3 channels by heterotrimeric G proteins. We have demonstrated the activation of Kir3.4 channels by agonist stimulation of non-PTX-sensitive G proteins under conditions of Gα subunit overexpression. We present evidence to suggest a key role for the coupling efficiency of Gα subunits in determining the specificity of Gβγ signaling to the channel.

Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K+ channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca2+-activated K+ channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K+. Thus, Ca2+-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices., Ion channels open and close to allow the regulated passage of ions through the membrane. Here the authors use selective ion channel blockers to analyse this regulation in a potassium channel and show that the gate is in the selectivity filter, past the entrance to the channel.

STARD4 (steroidogenic acute regulatory protein-related lipid-transfer domain containing 4) is known to transport cholesterol from the plasma membrane (PM) to intracellular organelles, such as the endoplasmic reticulum and endocytic recycling compartment, but it has not been known if the transport is selective. Identification of a surface-exposed basic region encompassing the C-terminal helix in the crystal structure of STARD4 suggested the possibility that anionic lipids confer membrane selectivity.

The determination of how ion channels open and close is crucial for understanding their physiological roles and for potential therapeutic modulation, but locating the gate within channels is not trivial. For voltage-gated Kv channels, a primary activation-gate formed by a helical bundle crossing near the intracellular pore entrance has been established. For several types of ligand-gated channels (CNG) and large-conductance Ca2+-activated K+ (BK) channels however, the K+ selectivity filter has been proposed to act as the conduction gate. We investigated the location of the voltage-dependent gate for the prokaryotic MthK channel, a BK channel homologue lacking voltage-sensor domains, using quaternary ammonium (QA) blockers as state-dependent probes. Intracellular QA blockers bind within an aqueous pore cavity between the putative intracellular-facing gate and the selectivity filter. Thus, these blockers ideally probe the gate location: an intracellular gate will only allow binding when open, whereas a selectivity filter gate will always allow binding. A kinetic analysis of tetrabutylammonium block of single MthK channels during gating determined that the voltage-dependent gate is located above the QA binding site in the pore. X-ray crystallographic analysis of the MthK pore with tetrabutylantimony confirmed QA binding immediately below the selectivity filter, unequivocally placing the voltage-dependent gate within the selectivity filter, akin to the C-type inactivation gate in eukaryotic K+ channels. In addition, state-dependent binding kinetics suggested that the selectivity filter inactivation led to conformational changes within the cavity and intracellular pore entrance, suggesting an allosteric connection between cytoplasmic domains and selectivity filter.

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl+) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H+). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is 0.25) in the bilayer-forming phospholipid mixture, alters KcsA H+ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H+ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.

Phosphoinositides are involved in cell-signaling pathways that regulate vital cell functions such as membrane excitability and trafficking, and cell metabolism, motility and proliferation. At the plasma membrane, phosphatidylinositol-4,5-bisphosphate (PIP2), which constitutes approximately 0.25% of cell phospholipid, is a key messenger in membrane-delimited signaling. PIP2 regulates structurally and functionally diverse membrane proteins including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters and receptors. The mechanism(s) by which PIP2 regulates many of its various “receptors” remain to be elucidated. Here we explore the notion that the amphiphilic phosphoinositides, by adsorbing to the bilayer/solution interface, alter bilayer properties such as curvature and elasticity. Such changes in bilayer properties can alter the equilibrium between membrane protein conformational states and thereby alter function. Taking advantage of the gramicidin channels’ sensitivity to changes in the lipid bilayer properties, we used fluorescence-based and single-channel gA assays to examine the effects of (diC8) phosphoinositides -PI, PI(4,5)P2, PI(3,5)P2, PI(3,4)P2 PI(3,4,5)P3 as well as long-chain PI(4,5)P2 on the lipid bilayer. The diC8 phosphoinositides, except for PI(3,5)P2, alter lipid bilayer properties with potency that decreases with increasing charge. Among the long-chain PI(4,5)P2s, the naturally occurring 1-stearyl-2-arachidonyl-PI(4,5)P2 is a more potent bilayer modifier than di-oleoyl-PI(4,5)P2. The diC8 and the naturally occurring PI(4,5)P2 have similar effects on short and long gA channels, indicating that changes in bilayer curvature dominate over those on bilayer elasticity. In contrast, diC8PI, which was more bilayer-active than diC8PIP2 altered bilayer elasticity. Our results show that application of exogenous PIP2 and its structural analogues (with changes in acyl chain length or phosphorylation state) alters lipid bilayer properties. These PIP2 lipid bilayer effects may be important for some of the many different effects on membrane protein function.

Acetylcholine signaling through muscarinic type 2 receptors activates atrial G protein-gated inwardly rectifying K(+) (Kir3) channels via the betagamma subunits of G proteins (Gbetagamma). Different combinations of recombinant Gbetagamma subunits have been shown to activate Kir3 channels in a similar manner. In native systems, however, only Gbetagamma subunits associated with the pertussis toxin-sensitive Galpha(i/o) subunits signal to K(+) channels. Additionally, in vitro binding experiments supported the notion that the C terminus of Kir3 channels interacts preferentially with Galpha(i) over Galpha(q). In this study we confirmed in two heterologous expression systems a preference of Galpha(i) over Galpha(q) in the activation of K(+) currents. To identify determinants of Gbetagamma signaling specificity, we first exchanged domains of Galpha(i) and Galpha(q) subunits responsible for receptor coupling selectivity and swapped their receptor coupling partners. Our results established that the G proteins, regardless of the receptor type to which they coupled, conferred specificity to Kir3 activation. We next tested signaling through chimeras between the Galpha(i) and Galpha(q) subunits in which the N terminus, the helical, or the GTPase domains of the Galpha subunits were exchanged. Our results revealed that the helical domain of Galpha(i) (residues 63-175) in the background of Galpha(q) could support Kir3 activation, whereas the reverse chimera could not. Moreover, the helical domain of the Galpha(i) subunit conferred "Galpha(i)-like" binding of the Kir3 C terminus to the Galpha(q) subunits that contained it. These results implicate the helical domain of Galpha(i) proteins as a critical determinant of Gbetagamma signaling specificity.

Atrial fibrillation (AF) is the most prevalent type of arrhythmia and is associated with significant mortality. Amiodarone, owing to its efficacy and minimal proarrhythmic effects, is the most commonly prescribed antiarrhythmic agent. Though amiodarone is considered a class III, repolarization-prolonging, antiarrhythmic that inhibits potassium channels involved in restoring the membrane excitable state, it also alters the function of many other structurally and functionally diverse membrane proteins. This concomitant regulation of multiple membrane proteins by amiodarone results in complex therapeutic and toxicity profiles. Other antiarrhythmics, such as dronedarone, have similar multiple membrane protein targets. Though such a multipronged mechanism for treating AF appears to be desired, it is not clear how amiodarone, and other antiarrhythmics exert their therapeutic actions and regulate a diverse range of membrane proteins at similar concentrations. Chatelane et al. (1989) found that amiodarone and propranolol alter lipid bilayer properties, and that amiodarone does so at therapeutic concentrations. We therefore took advantage of the gramicidin (gA) channels’ sensitivity to changes in the lipid bilayer properties to determine whether the commonly used antiarrhythmics amiodarone, dronedarone, propranolol and pindolol perturb the lipid bilayer properties and at which concentrations. Using a gA-based fluorescence assay and single-channel electrophysiology to explore the antiarrhythmics’ effects on the lipid bilayer, we found that amiodarone and dronedarone are potent bilayer modifiers, propranolol has intermediate activity, and pindolol is the least potent. Moreover, amiodarone and propranolol increase bilayer elasticity. Because amiodarone and dronedarone alter the lipid bilayer at their therapeutic concentrations, where they act on multiple membrane proteins, our results suggest that their multi-target effects may involve a lipid bilayer-mediated mechanism. This underscores the need to further explore the role of bilayer-mediated mechanism in therapeutic as well as toxic effects of antiarrhythmics agents.

Publisher Summary Tryptophan (Trp) analogs in proteins have great potential for fluorescence studies, provided they have a unique absorbance compared to Trp, allowing proteins incorporating these analogs to be selectively excited in the presence of other Trp-containing proteins. This chapter discusses attempts to replace the four Trps in the truncated, soluble domain of recombinant human tissue factor (sTF) with 5-hydroxytryptophan (5-OHTrp) and 7-azatryptophan (7-ATrp). Among possible Trp analogs, 7-ATrp is an excellent environmental probe, as it can exhibit a large increase in quantum yield, when buried in the protein interior and has approximately 50 nm red-shifted emission spectra compared to Trp, while the analog 5-OHTrp has the advantage of a large shift in the absorbance spectrum, allowing for its selective excitation in the presence of Trp. The chapter describes that cells exposed to 5-OHTrp grow poorly, and express less protein than cells induced in the presence of Trp. To date most of the proteins expressed with 5-OHTrp or 7-ATrp have been prokaryotic, and the prokaryotic DNA-binding proteins discussed have been prepared to provide absorption spectra that can be easily resolved from that of DNA. The other DNA-binding proteins all appear to have wild-type function. However, to date, there is not enough data available to make similar generalizations about eukaryotic proteins.

Complexation of the extracellular domain of tissue factor to the serine protease factor VIIa is a critical step in the process of blood coagulation following tissue damage. To study the structure and function of the extracellular domain of tissue factor (soluble tissue factor, sTF), we have used site-directed mutagenesis to replace each of sTF's four tryptophans (Trp) with phenylalanine (Phe) or tyrosine (Tyr). Replacement of any one of the four Trps reduced the protein stability against denaturation by guanidinium chloride in a similar manner, indicating that each residue has important structural interactions within the protein. Replacement of Trps 25, 45, and 158 resulted in reduced cofactor activities, indicating that these residues are located in regions important for biological activity. The activities of mutants with Trp 14 or both Trps 14 and 158 replaced were comparable to sTF. From the combination of absorbance and fluorescence spectra of the individual Trps, information is obtained showing that all the Trps are buried in the protein matrix, and Trps 14 and 25 are in highly constrained environments compared to Trps 45 and 158. To directly monitor interactions of sTF with factor VIIa and its substrate factor X, we have undertaken a program to generate spectrally enhanced protein (SEP) analogs of sTF and the sTF Trp mutants by in vivo incorporation of Trp analogs with absorbance and fluorescence distinct from Trp. Attempts to incorporate the Trp analogs 5-hydroxytryptophan (5-OHTrp) and 7-azatryptophan (7- ATrp) into sTF have provided further information on the structural significance of the Trp residues in sTF.

Thiazolidinediones (TZD) are selective peroxizome-proliferator receptor gamma (PPARγ) agonists that are used to treat hyperglycemia in type 2 diabetes. In addition to their hypoglycemic actions they have anti-inflammatory, anti-atherosclerotic and cardiovascular effects, but PPARγ activation does not account for all their actions. Three TZDs - troglitazone (Resulin), rosiglitazone (Avandia), and pioglitazone (Actos) - have been marketed; troglitazone was subsequently withdrawn due to hepatotoxicity and a precursor TZD - ciglitazone- was discontinued after phase II trials. TZDs, with troglitazone being the most potent, modulate L-type calcium and delayed-rectifier potassium channels by a seemingly PPARγ-independent mechanism. This could result from the adsorption of the amphiphilic TZDs to the membrane/solution interface, which can alter bilayer properties such as thickness, intrinsic curvature and the elastic moduli, and thus membrane protein function. We therefore examined whether TZDs alter lipid bilayer properties. We exploited the sensitivity of gramicidin channels to changes in bilayer properties to test for TZD-induced bilayer effects. TZDs alter gramicidin channel function and shift the monomer-dimer equilibrium toward the conducting dimers. Using gramicidin channels of different lengths we find that the TZD effects do not vary with changes in hydrophobic mismatch. Increasing bilayer stiffness with cholesterol amplifies the TZD-mediated changes in gramicidin channel function. Based on the concentrations at which we observe changes in gramicidin lifetime and appearance frequency, the potency is troglitazone>rosiglitazone>ciglitazone>pioglitazone, consistent with their effects on native membrane proteins. We examined the TZDs effects in native membranes using neuronal voltage-gated sodium channels (NaV) using whole-cell recordings. All TZDs caused a negative shift in the voltage-dependence of inactivation at concentrations similar to those that alter gramicidin channel function. Our results show that TZDs affect bulk membrane properties at concentrations that modulate native ion channels.

The KAch channel slows the heart rate it in response to acetylcholine (ACh). Binding of ACh to the M2 Muscarinic receptor triggers Gβγ-mediated activation of the cardiac GIRK1/GIRK4 inwardly rectifying K channel, which is a heterotetrameric complex of the Kir3.1 and Kir3.4 subunits. Several reports, including work from our laboratory, suggest that phosphorylation events may be critical determinants of the above regulation. Apart from the heterologous expression of the channel subunits in various systems, primary atrial cultures have been so far the only available system for such studies. The development of a cardiomyocyte cell line (HL-1) which has the ability to continuously divide while maintaining a differentiated cardiac phenotype, prompted us to examine whether it could be used for studies on the regulation of GIRK1/GIRK4 channel activity. Here we report that HL-1 cells express both the Kir3.1 and Kir3.4 subunits, antibodies raised against each subunit co-immunoprecipitate the other subunit, the cells respond to ACh and growth factor receptor stimulation, show KAch currents and display electrophysiological properties characteristic of atrial KAch. Our data indicate that the HL-1 cell line provides a useful tool for the dissection of mechanisms regulating GIRK1/GIRK4 activity in vivo.

The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins.

Functional studies of purified ion channels reconstituted in a defined membrane environment usually are conducted using single-channel electrophysiology. Ensemble-averaged methods are used infrequently, compared to single-channel electrophysiology because of the technical challenges involved in giant unilamellar liposome preparation and patching−though these methods yield important information that may be difficult to obtain from single-channel studies, e.g. (Chakrapani et al., 2007). To overcome some of the limitations encountered, we developed a sequential-push fluorescence-based stopped-flow assay to characterize channel function, using KcsA as the prototype. The method is based on earlier studies (Moore and Raftery 1980; Wu et al., 1981; Ingolfsson and Andersen), where channel activity was determined using the KcsA-permeable ANTS quencher, thallium (Tl+). We evaluate KcsA function from the rate of ANTS quenching in sequential-push stopped-flow measurements on the millisecond timescale. Detergent was removed using BioBeads, which minimizes ANTS consumption. To illustrate the method, we determined the KcsA activation by extravesicular H+ for WT KcsA and the non-inactivating E71A mutant channels. Our results demonstrate efficient and reproducible reconstitution. Fitting the activation curve using the Hill equation yields a pH for half-activation (pH0.5) similar to that obtained from single-channel electrophysiology for the E71A mutant (Thompson et al., 2008) and higher than that reported for WT KcsA (Chakrapani et al.). The Hill coefficients, n, were lower than those obtained with single-channel recording for E71A mutant but higher for WT KcsA. Our results suggest that both pH0.5 and n vary with changes in lipid bilayer thickness and bulk properties such as elasticity. The ease of vesicle preparation, cost effectiveness, reproducibility and time resolution makes this a powerful assay to explore KcsA activation and inactivation, and how they vary with changes in the channels' lipid bilayer environment.

Thiazolidinediones are widely used to treat hyperglycemia in patients suffering from type 2 diabetes. Three thiazolinediones - troglitazone (Resulin), rosiglitazone (Avandia), and pioglitazone (Actos) - have been marketed; troglitazone was subsequently withdrawn due to hepatotoxicity. The thiazolidinediones are selective peroxizome-proliferator receptor gamma (PPARγ) agonists and they increase insulin sensitivity. They also have been found to have anti-oxidant, anti-inflammatory, anti-atherosclerotic and cardiovascular effects, but PPARγ activation alone does not account for all their actions. All three derivatives, with troglitazone being the most potent, modulate L-type calcium and delayed-rectifier potassium Kv1.3 channels by a seemingly PPARγ-independent mechanism. This could result from the adsorption of amphiphilic molecules to the membrane, which can alter bilayer properties such as thickness, intrinsic curvature and elastic moduli, and thus membrane protein function. We therefore set out to determine whether the amphiphilic troglitazone and rosiglitazone alter lipid bilayer properties. Using gramicidin channels as probes, where we monitor the changes in channel lifetime and rate of appearance, we tested and compared the effects of troglitazone and rosiglitazone on channels of different lengths in DOPC bilayers. Troglitazone or rosiglitazone did not alter gramicidin channel conductances, suggesting that direct interactions are not involved. In contrast, the lifetimes of both channels increased with similar relative changes for both the shorter and the longer channels. Consistent with their effects on calcium and potassium channels troglitazone is more potent than rosiglitazone. Our results show that both troglitazone and rosiglitazone affect bulk membrane properties at the concentrations where they modulate other ion channels.