Molecular Mechanism of TMEM16A CA2+-Activated Chloride Channel Desensitization

Monday, February 13, 2017 activator of Kir(Inwardly-rectifying Kþ) channels. Here we show that hydrogen sulfide(H2S), an emerging gasotransmitter, produced in cells, regulates the activity of several members of the Kir channel family differentially via s-sulfhydration of its cytoplasmic cysteine residues and the extent of regulation depends on the strength of channel-PIP2 interaction. These findings have used two independent methods to demonstrate H2S effects: direct administration of an exogenous donor, sodium hydrogen sulfide(NaHS), and expression of cystathionine gamma lyase(CSE), the principle producer of endogenous H2S in peripheral tissues, such as in the cardiovascular system and specific regions of the brain. We show here that H2S regulation depends on the strength of channel-PIP2 interactions by employing techniques that alter the extent of channel-PIP2 interactions. H2S regulation is attenuated when strengthening channel-PIP2 interactions either through point mutations in Kir3.2(altering channel structure intrinsically), interacting proteins(e.g. G-beta-gamma) or by increasing intracellular PIP2 levels by coexpression of PIP5Kinase(PIP5K), the enzyme that converts endogenous PIP(4) into PIP2(4,5). Conversely, our results show increased H2S regulation when channel-PIP2 interactions are weakened through the administration of Wortmannin(Wort), a known blocker of PIP4K, which leads to abatement of intracellular PIP2. In addition, activation of co-expressed Ci-VSP, a voltage activated PIP5 phosphatase, also reduces intracellular PIP2, and H2S is able to exert greater effects on the channel. These H2S effects depend on the presence of specific cytoplasmic cysteine residues in Kir3.2(GIRK2), as their replacement(by other non-polar amino acids) abolishes the H2S effect(without affecting channel-PIP2 interactions). Reintroduction of specific cysteine residues back in to the background of this GIRK2 cysteine less mutant rescues the H2S effect. In order to monitor H2S modification, we employ the biochemical technique, Tag Switch Assay(TSA), showing that Kir3.2 is directly s-sulfhydrated at specific cysteine residues upon addition of exogenous H2S via administration of NaHS to wild-type Kir3.2 and Kir3.2 mutants lacking specific cysteine residues. Lastly, molecular dynamics simulation experiments are providing mechanistic insight in how s-sulfhydration of these specific cysteine residues would lead to changes in affecting channel-PIP2 interactions and channel gating. These studies reveal for the first time the intricate interplay between Kir channels(proteins), PIP2(biological lipids) and H2S(gaseous molecules). 1122-Pos Board B190 General Anesthetics Minimally affect Lipid Bilayer Properties at Clinical Concentrations Karl Herold1, William Lee1, R.L. Sanford2, Olaf S. Andersen2, Hugh C. Hemmings Jr1. 1 Anesthesiology, Weill Cornell Medicine, New York, NY, USA, 2 Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, USA. General anesthetics are widely used but their exact molecular mechanisms are incompletely understood. One early model, known as the unitary lipid-based hypothesis of anesthetic action, proposed that nonspecific anesthetic interactions with the lipid bilayer altered neuronal function through effects on membrane properties. This model, which is based largely on the Meyer-Overton correlation between anesthetic potency and lipid solubility, has been challenged by more recent evidence supporting direct interactions of anesthetics with a range of proteins, in particular transmembrane ion channels. The available results, however, do not exclude anesthetic modulation of ion channel function through interactions with the lipid bilayer. We therefore tested the effects of a range of chemically diverse general anesthetics (diethyl ether, desflurane, sevoflurane, isoflurane, halothane, chloroform, bromoform, cyclopropane, F3, ketamine, etomidate and propofol) and related nonanesthetics (F6 and flurothyl, predicted to be anesthetic based on their lipid solubilities) on lipid bilayer properties using a sensitive gramicidin-based assay. We show that none of the anesthetics or nonanesthetics tested significantly altered lipid bilayer properties at clinically relevant concentrations. Even at supra-anesthetic concentrations, most anesthetics produced only minimal bilayer-mediated effects, though some were able to alter lipid bilayer properties at supra-therapeutic (toxic) concentrations. We conclude that the effects of general anesthetics at clinically relevant concentrations involve minimal changes in lipid bilayer properties. Therefore clinical anesthesia is likely to result from direct interactions of anesthetics with membrane proteins. Supported by GM058055 (HCH) and GM021347 (OSA). 1123-Pos Board B191 Lipid Profile and Functionality of Nicotinic Aceytilcholine Receptor from Torpedo Californica Solubillized with Cyclofos Detergent Family Orestes Quesada1, Jose´ O. Colon-Sa´ez2, Carol L. Gonza´lez3, Carol L. Gonza´lez3, Rafael Maldonado4, Irvin Rosado4, Alan Espinal4, Jesus Acevedo4, Jose´ A. Lasalde-Dominicci1.

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Physical Sciences, University of Puerto Rico, San Juan, San Juan, PR, USA, Department of Pharmaceutical Sciences, School of Pharmacy, University of Puerto Rico, Medical Sciences Campus, San Juan, PR, USA, 3Department of Chemistry, University of Puerto Rico, San Juan, San Juan, PR, USA, 4 Biology, University of Puerto Rico, San Juan, San Juan, PR, USA. The first step for reconstitution in attempts to membrane protein crystallization is the solubilization process by detergents. During this process, preservation of the native protein structure and adjacent lipid environments is crucial for activity and stability. The detergent disruption of lipid-lipid, lipid-protein interactions alter the aggregation state, stability, and functionality of the native structure. The effect is more severe when protein activity is lipid dependent. The molecular composition of all detected phospholipid classes from affinity-purified acetylcholine receptors detergent complexes (nAChR-DCs) from Torpedo californica (Tc) solubilized using a homologous series of Cyclo Foscholine (CF) detergents were analyzed by Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). We took advantage of Xenopus laevis oocytes expression system to access the nAChR-DCs activity by means of two electrode voltage clamp (TEVC) technique. UPLC/ESI/MS/MS results shown a phospholipid composition favored by phosphatidylcholine and sphingomyelin species for the three nAChR-DC studied. However, the molecular species in each nAChr-DC and cholesterol content were in some degree, quite different despite that the only detergent structural difference is 1 or 2 methylene groups. Injection of oocytes with Tc nAChRs-DCs solubilized with CF family, differing in the length of their acyl chain, resulted in responses with amplitudes that inversely depend on acyl chain length. CF-4 displayed the highest mean amplitudes in this family (200 5 60 nA) which is about 80% of the current produced when normalized to the mean current of the crude membrane. The longer side chain, CF-6 and CF-7 produced an insignificant current compared to the crude membrane. These findings will provide a lipidomic strategy to prepare nAChR-DCs suitable for structural studies. 2

1124-Pos Board B192 Cu2D Ions Modulate the Conductance Hysteresis of Lysenin Channels Devon Richtsmeier1, Nisha Shrestha2, Sheenah Bryant2, Christopher A. Thomas1, Andy Bogard1, Charles Hanna2, Daniel Fologea2. 1 Department of Physics, Boise State University, Boise, ID, USA, 2 Department of Physics/Biomolecular Sciences Graduate Program, Boise State University, Boise, ID, USA. Lysenin channels self-assembled into lipid membranes have unique biophysical features which distinguish them from other pore-forming toxins. Lysenin channels present hysteretic and asymmetrical voltage-induced gating in response to positive transmembrane voltages and reversible ligand-gating in the presence of multivalent ions. Previous work on lysenin channels reported no apparent dependency between the voltage and ligand gating mechanisms. Such a link is demonstrated by this work, where we show that Cu2þ ions, known to be very potent inhibitors of lysenin channels’ conductance, have a dramatic influence on the channel response to transmembrane voltages. Addition of Cu2þ ions (in the range 0-100 mM) to the solutions bathing a population of lysenin channels inserted into a planar lipid membrane induces the well-known conductance inhibition at any transmembrane voltage within the physiological range. However, the channels that remain open respond to positive voltages by closing. Unexpectedly, only minor changes of the midway voltage of activation and the open probability are encountered, which is indicative of a lack of strong ionic screening induced by Cu2þ ions. In the absence of Cu2þ ions, channel reopening, although it occurs at lower voltages than closing (indicative of hysteresis), is always complete at voltages lower than þ10 mV. In the presence of Cu2þ ions, channel reopening is not complete until the voltages reach values as low as 40 mV. This channel arresting in the closed state is concentration dependent and manifests as dramatic changes in the opening and closing rate constants in the presence of Cu2þ ions. The presence of Cu2þ ions significantly accentuate the hysteretic behavior of lysenin channels, indicative of improved molecular memory capabilities. Given the link between hysteresis and memory, our work may open novel avenues for exploiting the voltage-controlled molecular memory for bioelectronics and biological interfacing. 1125-Pos Board B193 Molecular Mechanism of TMEM16A CA2D-Activated Chloride Channel Desensitization Son C. Le. Biochemistry, Duke University, Durham, NC, USA. TMEM16A forms the long-sought after calcium-activated chloride channel (CaCC) that is highly expressed in various tissues such as dorsal root

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ganglia (DRGs), epithelial and smooth muscle cells. Extensive studies have demonstrated that the TMEM16A-CaCC plays important roles in mediating nociception, trans-epithelial chloride transport and smooth muscle contraction. Whilst the activation mechanism of the TMEM16A-CaCC has been extensively explored, how the channel activity is regulated in different cell types remains elusive. By studying the molecular mechanism of TMEM16A-CaCC desensitization, a well-documented characteristic of CaCCs, we aim to understand how the TMEM16A-CaCC is regulated at molecular and cellular levels. Our findings will not only illustrate the desensitization mechanism of the TMEM16A-CaCC channel, but add new insights on understanding the channel activation mechanism. As the desensitization severely hampers the biophysical characterization of TMEM16A-CaCC properties, our findings will provide a solution to prevent CaCC desensitization and thus facilitate the structure-function study of the TMEM16 channels. 1126-Pos Board B194 Competitive Interactions of PIRT and PI(4,5)P2 Modulate TRPM8 Nicholas Sisco, Parthasarathi Rath, Jacob K. Hilton, Cole V.M. Helsell, Wade D. Van Horn. Arizona State University, Tempe, AZ, USA. PIRT or Phosphoinositide regulator of TRP is a small membrane protein that has been shown to modulate the function of TRP channels. PIRT is implicated in fine-tuning the physiological roles that TRP channels play in thermosensing and ligand activation; suggesting roles for PIRT:TRP complexes in pathophysiologies such as pruritus and chronic pain. In an effort to better understand the mechanism of PIRT modulation of TRP channels, human PIRT has been expressed and purified. Solution NMR studies of the resulting protein have given way to backbone resonance assignment allowing for experimental determination of its secondary structure and membrane topology. PIRT contains a relatively unstructured N-terminus with two transmembrane helices followed by a positively charged PI(4,5)P2 interacting site in the juxtamembrane region near the C-terminus. Further residue specific characterization of PIRT interactions detail the residue specific binding of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and the TRPM8 S1-S4 sensing domain. Interestingly, PIRT residues that specifically bind PI(4,5)P2 also specifically interact with the TRPM8 sensing domain. The overlap in PIRT binding sites between TRPM8 and PI(4,5)P2 suggest a lipid shuttling mechanism between TRPM8 and PIRT as part of the regulatory function. These studies provide the first glimpses of a structural and molecular framework to begin to dissect the regulatory mechanism of the tripartite PIRT–TRPM8–PI(4,5) P2 complex. 1127-Pos Board B195 Identification of Residues Important for Ion and Lipid Transport in a TMEM16 Scramblase Byoung-Cheol Lee1, Maria Falzone2, Anant Menon3, Alessio Accardi4. 1 Anesthesiology, Weill Cornell Medical College, New York, NY, USA, 2 Graduate School, Weill Cornell Medical College, New York, NY, USA, 3 Biochemistry, Weill Cornell Medical College, New York, NY, USA, 4 Anesthesiology, Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA. The TMEM16 family comprises Ca2þ-activated Cl- channels and phospholipid scramblases. The recent crystal structure of a fungal homologue, nhTMEM16, revealed an important architectural feature of this protein family in the form of a bilayer-spanning hydrophilic groove that is directly exposed to the membrane and provides a possible pathway for lipid translocation. Mutations that alter ion channel activity of the TMEM16 proteins localize around the groove, suggesting that the ion and lipid pathways coincide. However, an initial report suggested that nhTMEM16, unlike other TMEM16 scramblases, does not mediate ion transport. Here, we show that nhTMEM16 mediates simultaneous ion and lipid transport and that its properties closely resemble those of afTMEM16.To test the hypothesis the hydrophilic groove forms a shared pathway for ions and lipids we mutated residues facing the groove to tryptophan (Trp), as its bulky and hydrophobic side chain should impair transport. We then investigated how these mutations affect scrambling and ion transport. Of the 17 Trpmutants tested, 40% had little to no effect on scrambling (<25% reduction), 40% had intermediate effects (25 to 50% reduction) while only 20% had high impact (>50% reduction). Interestingly, the impact on ion transport closely mirrored that on scrambling, consistent with the idea that these two functions are tightly coupled. We identify a set of 3 residues which

define a constriction in the groove, which when mutated to Trp cause a dramatic reduction in the rate of scrambling. Unexpectedly however, this effect is independent of the size of the introduced side chain as mutations to Alanine (Ala) has a comparably adverse impact on scrambling. Taken together our results indicate that the specific chemico-physical properties of these 3 residues lining the groove is crucial to lipid and ion transport of nhTMEM16.

General Protein-Lipid Interactions II 1128-Pos Board B196 Human SP-A1 Enhances Interfacial Properties of Lung Surfactant and Restores a Proper Behavior in the Presence of Inhibitory Agents Raquel Arroyo1, Elena Lopez-Rodriguez1,2, Alicia Pascual1, Mercedes Echaide1, Joanna Floros3, Jesus Perez-Gil4. 1 Biochemistry & Molecular Biology I, Complutense University of Madrid, MADRID, Spain, 2Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany, 3Obstetrics and Gynecology, Penn State College of Medicine, Hershey, PA, USA, 4Biochemistry & Molecular Biology I / Research Institute Hospital 12 de Octubre, Complutense University of Madrid, MADRID, Spain. Pulmonary surfactant is a lipid-protein complex secreted by type II alveolar cells. This surface-active agent prevents alveolar collapse by reducing surface tension at the respiratory air-liquid interface during expiration and contributes to the innate defense of the lung. Surfactant protein A (SP-A) is a hydrophilic collectin protein implicated mainly in the host defense. However, it has been shown that SP-A participates in the formation of surfactant films at the interface and helps to reestablish the biophysical properties of surfactant when this is impaired by the presence of some inhibitors. There are two SP-A genes in humans, SFTPA1 and SFTPA2, which products differ in structure and function. With the aim of investigating the differences in the biophysical properties of surfactant containing human SP-A1, SP-A2 or both, we have studied 1) pulmonary surfactant from individual humanized transgenic mice expressing human SP-A1, SP-A2, or both SP-A1/2, in a Captive Bubble Surfactometer (CBS), and 2) the capability of this two variants of the protein to restore the biophysical activity of a reconstituted organic extract (OE) from native porcine surfactant in the presence of serum as inhibitory agent, mimicking a situation of lung injury. The experiments revealed consistent functional differences in surfactant containing SP-A1 or SP-A2. In the presence of serum, surfactant from mice containing SP-A1 and the OE reconstituted upon addition of SPA1, achieved lower surface tension after post-expansion interfacial adsorption and a more efficient reorganization of the film under interfacial compressionexpansion cycling conditions, than surfactants containing no SP-A or only SP-A2. We conclude that the presence of hSP-A1 allows surfactant to adopt a particularly favorable structure with optimal biophysical properties and helps to restore a proper behavior in the presence of an inhibitory agent as serum. 1129-Pos Board B197 The Relationship of Sequence and Activity in a Series of Short b-Sheet Peptides with Antimicrobial Activity Samantha Kennelly1, Larry Masterson2, Jalen Hoehn1, Andrew Hexum1. 1 Hamline University, St Paul, MN, USA, 2Hamline University, Saint Paul, MN, USA. Infectious diseases are the second-leading cause of death worldwide, with a declining potency of current antibiotics due to the increased drug resistance of bacteria. Due to this phenomenon, antimicrobial peptides (AMPs) have gained a wide interest due to their potential in solving the antibioticresistance problem. AMPs present specificity for bacterial cells and disrupt their membranes, eventually leading to the death of the cell. In this research study, a series of peptides that were previously reported to have antimicrobial activity- (IRIR)2, (IRIK)2 and (VRVK)2-were investigated using model lipid membranes. Small unilamellar vesicles containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) head groups were used to mimic various cell eukaryotic and bacterial membranes to evaluate their ability to recognize and disrupt these membranes. Solution turbidity measurements were used to measure aggregation, while fluorescence resonance energy transfer (FRET) experiments were used to measure membrane fusion, both indicating that the peptides function act via the carpet model mechanism. The lytic activity of the peptides was then verified using a calcein leakage assay. This experiment illustrated that the level of activity for (IRIR)2 is much greater than that of both