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TMEM16A Mediates the Fast Block to Polyspermy in Xenopus Laevis Eggs
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Wednesday, February 15, 2017
design represent the actual innovation gap, especially against Gram-negative bacteria. These are particularly challenging due to their outer membrane, which works to modulate the entry of chemical species under strict control. Among the possible strategies to overcome this barrier, siderophores suggest an intriguing one, the Trojan horse strategy. Bacteria make use of endogenous/exogenous chelating agents, the siderophores, to capture Fe3þ from the environment. Complexes are then retrieved by specific outer membrane receptors that work within multi-proteins machineries. By connecting/ mimicking an antibiotic to a siderophore it is possible to trick bacterial defenses. Despite considerable efforts expended in both natural and synthetic conjugates, only few compounds have met with success. As crystal structures of siderophore receptors are becoming available, increased understanding of siderophore-metal complex recognition will certainly help to develop new siderophores and linkages with improved performance. BAL30072 is an interesting monocyclic b-lactam antibiotic belonging to the sulfactams. It was designed from tigemonam by introducing an iron chelating 1,3-dihydroxy-4-pyridinone group. It shows remarkable activity against important Gram-negative pathogens, including multidrug-resistant Pseudomonas aeruginosa and Acinetobacter sp. isolates. In this work, BAL30072 and several analogues have been compared in terms of metal-complex formation in different environments, namely, dimethylsulfoxide and water at different pH. NMR titrations have been performed with Ga3þ ions in order to retrieve stoichiometry and relative stability of the complexes. Inter-proton distances were also determined in some instances and the corresponding molecular model formulated. The experimental data have been complemented by a combination of quantum and classical MD simulations to elucidate structural and dynamical properties of compounds. Molecular docking was then performed to investigate the binding to the putative BAL30072 receptor in P. aeruginosa, PiuA. 2719-Pos Board B326 TMEM16A Mediates the Fast Block to Polyspermy in Xenopus Laevis Eggs Katherine L. Wozniak, Brianna L. Mayfield, Anne E. Carlson. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA. Ca2þ-activated Cl- channels (CaCCs) mediate some of the most important physiologic processes, such as the regulation of neuronal and cardiac excitability, electrolyte balances in secretory epithelia, and olfactory transduction. However, it wasn’t until 2008 that transmembrane proteins with unknown function 16 (TMEM16a) was identified as a bona fide CaCC. One of the cell types that TMEM16a was originally identified in was Xenopus laevis oocytes. Although TMEM16a is present in these oocytes, we do not know if it is maintained during oogenesis and functional in the fertilization-competent egg. During fertilization in X. laevis, a Ca2þ-activated Cl- current is evoked in a pathway known as the fast polyspermy block, which inhibits sperm from entering an already fertilized egg. Here, we sought to uncover the molecular identity of the Cl- channel mediating this polyspermy block. We tested whether the rate of the fertilization-evoked depolarization was altered in eggs inseminated in the presence of TMEM16a-targeting small molecule inhibitors including: MONNA, Ani9, CaCCinh-A01, and T16ainh-A01. We reasoned that the rate of depolarization is proportional to the number of channels opened and thus looked for changes in the depolarization rate. We found that the depolarization of the fast block was significantly slowed or completely prevented in eggs inseminated in the presence of these TMEM16a inhibitors. To verify that the TMEM16a-targeted inhibitors block the X. laevis version of the channel, we recorded the Ca2þ activated Cl- current evoked by uncaging IP3 to evoke Ca2þ release from the endoplasmic reticulum, in X. laevis oocytes using two electrode voltage clamp. We found that these inhibitors indeed were effective on the X. laevis TMEM16a channels. Taken together, these results suggest that TMEM16a is conserved through the oogenesis process and that TMEM16a mediates the fast block to polyspermy in X. laevis.
Ligand-gated Channels II 2720-Pos Board B327 Functional Characterization of a Nicotinic Acetylcholine Receptor from an Extremophile Worm Eveline Wijckmans, Florian Delbart, Chris Ulens. Laboratory of Structural Neurobiology, KU Leuven, Leuven, Belgium. Cys-loop receptors (CLRs) or pentameric ligand-gated ion channels (pLGICs) are a class of integral membrane proteins involved in fast excitatory or inhibitory synaptic neurotransmission. Known representatives of the family are the nicotinic acetylcholine receptor (nAChR), the serotonin-3 receptor (5-HT3R), the glycine receptor (GlyR) and the subtype
A/C g-aminobutyric acid receptor (GABAA/CR). Over the last years, important structural information emerged with the structure determination of the b3 GABAR, the 5-HT3AR, the a1 and a3 GlyR and the a4b2 nAChR. Despite this tremendous progress crystal structures of integral homopentameric nAChRs are still lacking. Previously, we identified a putative nAChR in the genome of the extreme thermophile worm Alvinella pompejana, named Alpo4, which we can express and purify in sufficient amounts for structural studies. Following solubilization with a variety of detergents we observed that Alpo4 is biochemically stable and assembles into pentamers as observed with electron microscopy. In this study, we further characterize the functional properties of Alpo4 after reconstitution of purified protein in planar lipid bilayers and subsequent patch clamp electrophysiology. Our recordings indicate that Alpo4 can be activated by the agonists nicotine or acetylcholine. Furthermore, currents can be modulated by allosteric modulators such as ivermectin or PNU-120596 and subsequently blocked by a pore blocker such as memantine or a competitive antagonist such as a-bungarotoxin. This pharmacological profile suggests that Alpo4 is a representative member of the nAChR subfamily. Ongoing experiments aim to quantify the single channel properties of Alpo4. Collectively, the results from our functional and biochemical studies suggest that Alpo4 is a suitable candidate for future structure determination of an integral homopentameric nAChR. 2721-Pos Board B328 Acetylcholine Receptor Gating: Click-Twist-Tilt-Rip-Pop Shaweta Gupta, Srirupa Chakraborty, Ridhima Vij, Tapan K. Nayak, Anthony Auerbach. Biophysics, University at Buffalo, Buffalo, NY, USA. We propose a testable model for AChR C4O gating that is consistent with the following. i) Mutational energy changes are mostly local. Mutant-cycle ˚ do not interact, analyses show that most side chains separated by >15A except for the aM2M3 linker that interacts significantly with residues at the binding sites. ii) Gating is a sequential process. Phi gives the free energy character (structure) of an amino acid at the gating transition state on a scale from 1 (O-like) to 0 (C-like). Phi values are distributed as 5 Gaussians and decrease domain-wise binding sites to gate, except for the aM2M3 linker that has the same phi as the binding sites. iii) There is a transition state ensemble (TSE). The number and relative heights of energy barriers in the TSE can be computed from phi values. There are 4 short-lived intermediate states, and opening is uphill. iv) It takes time to cross the TSE. We assume a ~5 ms shut component in single-channel recordings (‘flip/primed’) reflects sojourns in the TSE. Simulations suggest that the lifetime of each TSE intermediate is ~1 ms. v) C and O are stable. The opening/closing rate constants of WT adult AChRs are (ms1) ~50/2 with 2 bound ACh and ~106/10 without any agonists. vi) Structures. There are locally-closed GLIC structures. In opening the extracellular domain (ECD) rotates/compacts, membrane helices move and the gate unpacks. In our model, opening starts with coupled fluctuations at the aM2M3 linker and the transmitter-binding sites (click), followed by rotation of the ECD (twist), rearrangement of the helices (tilt), dilation of the gate (rip) to form a bubble that collapses to initiate ion flow (pop). Closing is the reverse and starts with bubble formation (the main structural correlate of ‘flip’). The most energetically-unfavorable step in opening is twisting. 2722-Pos Board B329 A Fluorescent Agonist of the Muscle Nicotinic Acetylcholine Receptor Abhilasha Ladha1, Vera Martos2, Andrew Plested3, Jana Kusch1. 1 Institute of Physiology II, University Hospital Jena, Jena, Germany, 2 Medicinal Chemistry, Leibniz Institute of Molecular Pharmacology, Berlin, Germany, 3Molecular Neuroscience and Biophysics, Leibniz Institute of Molecular Pharmacology, Berlin, Germany. The muscle-type nicotinic acetylcholine receptor (nAChR) is expressed at the neuromuscular junction, where it mediates the fast synaptic response upon ACh release from a motoneuron. It is one of the most studied ion channels and the gating mechanism has been described intensively. However, the reciprocal relationship between activation state and agonist binding is still elusive. Herein, we adapted a challenging approach, the so-called confocal patch-clamp fluorometry (cPCF) (Biskup et al., Nature 2007, 446:440-3) to study state-dependent agonist binding and unbinding behavior in nAChRs. We introduce a novel fluorescently tagged ACh derivative, fACh, which is an excellent congener of the native neurotransmitter agonist for at adult muscle-type nAChRs. For whole-cell recordings, HEK cells transiently transfected with muscle nAChRs were lifted and positioned in front of a double-barreled application pipette. Fast solution exchange was realized by a piezo-driven device. fACh was found to be similarly
Wednesday, February 15, 2017
design represent the actual innovation gap, especially against Gram-negative bacteria. These are particularly challenging due to their outer membrane, which works to modulate the entry of chemical species under strict control. Among the possible strategies to overcome this barrier, siderophores suggest an intriguing one, the Trojan horse strategy. Bacteria make use of endogenous/exogenous chelating agents, the siderophores, to capture Fe3þ from the environment. Complexes are then retrieved by specific outer membrane receptors that work within multi-proteins machineries. By connecting/ mimicking an antibiotic to a siderophore it is possible to trick bacterial defenses. Despite considerable efforts expended in both natural and synthetic conjugates, only few compounds have met with success. As crystal structures of siderophore receptors are becoming available, increased understanding of siderophore-metal complex recognition will certainly help to develop new siderophores and linkages with improved performance. BAL30072 is an interesting monocyclic b-lactam antibiotic belonging to the sulfactams. It was designed from tigemonam by introducing an iron chelating 1,3-dihydroxy-4-pyridinone group. It shows remarkable activity against important Gram-negative pathogens, including multidrug-resistant Pseudomonas aeruginosa and Acinetobacter sp. isolates. In this work, BAL30072 and several analogues have been compared in terms of metal-complex formation in different environments, namely, dimethylsulfoxide and water at different pH. NMR titrations have been performed with Ga3þ ions in order to retrieve stoichiometry and relative stability of the complexes. Inter-proton distances were also determined in some instances and the corresponding molecular model formulated. The experimental data have been complemented by a combination of quantum and classical MD simulations to elucidate structural and dynamical properties of compounds. Molecular docking was then performed to investigate the binding to the putative BAL30072 receptor in P. aeruginosa, PiuA. 2719-Pos Board B326 TMEM16A Mediates the Fast Block to Polyspermy in Xenopus Laevis Eggs Katherine L. Wozniak, Brianna L. Mayfield, Anne E. Carlson. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA. Ca2þ-activated Cl- channels (CaCCs) mediate some of the most important physiologic processes, such as the regulation of neuronal and cardiac excitability, electrolyte balances in secretory epithelia, and olfactory transduction. However, it wasn’t until 2008 that transmembrane proteins with unknown function 16 (TMEM16a) was identified as a bona fide CaCC. One of the cell types that TMEM16a was originally identified in was Xenopus laevis oocytes. Although TMEM16a is present in these oocytes, we do not know if it is maintained during oogenesis and functional in the fertilization-competent egg. During fertilization in X. laevis, a Ca2þ-activated Cl- current is evoked in a pathway known as the fast polyspermy block, which inhibits sperm from entering an already fertilized egg. Here, we sought to uncover the molecular identity of the Cl- channel mediating this polyspermy block. We tested whether the rate of the fertilization-evoked depolarization was altered in eggs inseminated in the presence of TMEM16a-targeting small molecule inhibitors including: MONNA, Ani9, CaCCinh-A01, and T16ainh-A01. We reasoned that the rate of depolarization is proportional to the number of channels opened and thus looked for changes in the depolarization rate. We found that the depolarization of the fast block was significantly slowed or completely prevented in eggs inseminated in the presence of these TMEM16a inhibitors. To verify that the TMEM16a-targeted inhibitors block the X. laevis version of the channel, we recorded the Ca2þ activated Cl- current evoked by uncaging IP3 to evoke Ca2þ release from the endoplasmic reticulum, in X. laevis oocytes using two electrode voltage clamp. We found that these inhibitors indeed were effective on the X. laevis TMEM16a channels. Taken together, these results suggest that TMEM16a is conserved through the oogenesis process and that TMEM16a mediates the fast block to polyspermy in X. laevis.
Ligand-gated Channels II 2720-Pos Board B327 Functional Characterization of a Nicotinic Acetylcholine Receptor from an Extremophile Worm Eveline Wijckmans, Florian Delbart, Chris Ulens. Laboratory of Structural Neurobiology, KU Leuven, Leuven, Belgium. Cys-loop receptors (CLRs) or pentameric ligand-gated ion channels (pLGICs) are a class of integral membrane proteins involved in fast excitatory or inhibitory synaptic neurotransmission. Known representatives of the family are the nicotinic acetylcholine receptor (nAChR), the serotonin-3 receptor (5-HT3R), the glycine receptor (GlyR) and the subtype
A/C g-aminobutyric acid receptor (GABAA/CR). Over the last years, important structural information emerged with the structure determination of the b3 GABAR, the 5-HT3AR, the a1 and a3 GlyR and the a4b2 nAChR. Despite this tremendous progress crystal structures of integral homopentameric nAChRs are still lacking. Previously, we identified a putative nAChR in the genome of the extreme thermophile worm Alvinella pompejana, named Alpo4, which we can express and purify in sufficient amounts for structural studies. Following solubilization with a variety of detergents we observed that Alpo4 is biochemically stable and assembles into pentamers as observed with electron microscopy. In this study, we further characterize the functional properties of Alpo4 after reconstitution of purified protein in planar lipid bilayers and subsequent patch clamp electrophysiology. Our recordings indicate that Alpo4 can be activated by the agonists nicotine or acetylcholine. Furthermore, currents can be modulated by allosteric modulators such as ivermectin or PNU-120596 and subsequently blocked by a pore blocker such as memantine or a competitive antagonist such as a-bungarotoxin. This pharmacological profile suggests that Alpo4 is a representative member of the nAChR subfamily. Ongoing experiments aim to quantify the single channel properties of Alpo4. Collectively, the results from our functional and biochemical studies suggest that Alpo4 is a suitable candidate for future structure determination of an integral homopentameric nAChR. 2721-Pos Board B328 Acetylcholine Receptor Gating: Click-Twist-Tilt-Rip-Pop Shaweta Gupta, Srirupa Chakraborty, Ridhima Vij, Tapan K. Nayak, Anthony Auerbach. Biophysics, University at Buffalo, Buffalo, NY, USA. We propose a testable model for AChR C4O gating that is consistent with the following. i) Mutational energy changes are mostly local. Mutant-cycle ˚ do not interact, analyses show that most side chains separated by >15A except for the aM2M3 linker that interacts significantly with residues at the binding sites. ii) Gating is a sequential process. Phi gives the free energy character (structure) of an amino acid at the gating transition state on a scale from 1 (O-like) to 0 (C-like). Phi values are distributed as 5 Gaussians and decrease domain-wise binding sites to gate, except for the aM2M3 linker that has the same phi as the binding sites. iii) There is a transition state ensemble (TSE). The number and relative heights of energy barriers in the TSE can be computed from phi values. There are 4 short-lived intermediate states, and opening is uphill. iv) It takes time to cross the TSE. We assume a ~5 ms shut component in single-channel recordings (‘flip/primed’) reflects sojourns in the TSE. Simulations suggest that the lifetime of each TSE intermediate is ~1 ms. v) C and O are stable. The opening/closing rate constants of WT adult AChRs are (ms1) ~50/2 with 2 bound ACh and ~106/10 without any agonists. vi) Structures. There are locally-closed GLIC structures. In opening the extracellular domain (ECD) rotates/compacts, membrane helices move and the gate unpacks. In our model, opening starts with coupled fluctuations at the aM2M3 linker and the transmitter-binding sites (click), followed by rotation of the ECD (twist), rearrangement of the helices (tilt), dilation of the gate (rip) to form a bubble that collapses to initiate ion flow (pop). Closing is the reverse and starts with bubble formation (the main structural correlate of ‘flip’). The most energetically-unfavorable step in opening is twisting. 2722-Pos Board B329 A Fluorescent Agonist of the Muscle Nicotinic Acetylcholine Receptor Abhilasha Ladha1, Vera Martos2, Andrew Plested3, Jana Kusch1. 1 Institute of Physiology II, University Hospital Jena, Jena, Germany, 2 Medicinal Chemistry, Leibniz Institute of Molecular Pharmacology, Berlin, Germany, 3Molecular Neuroscience and Biophysics, Leibniz Institute of Molecular Pharmacology, Berlin, Germany. The muscle-type nicotinic acetylcholine receptor (nAChR) is expressed at the neuromuscular junction, where it mediates the fast synaptic response upon ACh release from a motoneuron. It is one of the most studied ion channels and the gating mechanism has been described intensively. However, the reciprocal relationship between activation state and agonist binding is still elusive. Herein, we adapted a challenging approach, the so-called confocal patch-clamp fluorometry (cPCF) (Biskup et al., Nature 2007, 446:440-3) to study state-dependent agonist binding and unbinding behavior in nAChRs. We introduce a novel fluorescently tagged ACh derivative, fACh, which is an excellent congener of the native neurotransmitter agonist for at adult muscle-type nAChRs. For whole-cell recordings, HEK cells transiently transfected with muscle nAChRs were lifted and positioned in front of a double-barreled application pipette. Fast solution exchange was realized by a piezo-driven device. fACh was found to be similarly