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The N-Terminal Helix Acts as a Dynamic Membrane Coupler in the Gating Cycle of the Mechanosensitive Channel MscL
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Sunday, February 28, 2016
into account for screening molecules with improved permeation properties in rational drug designing. 1. Nikaido, H. Microbiol Mol Biol Rev 67, 593-656 (2003). 2. Lou, H. et al. PLoS ONE 6, e25825 (2011). 3. A. Kumar et al. J Phys Chem B 114, 9608-9616 (2010). 4. S. Acosta-Gutierrez. J Phys Chem Lett 6, 1807-1812 (2015). 591-Pos Board B371 GsMTx4 Mutagenesis Reveals Dynamic Membrane Binding Properties that Confer Inhibition of Piezo1 Radhakrishnan Gnanasambandam1, Chiranjib Ghatak2, Anthony Yasmann3, Frederick Sachs1, Alexey S. Ladokhin2, Sergei I. Sukharev3, Thomas M. Suchyna1. 1 Department of Physiology and Biophysics, SUNY University at Buffalo, Buffalo, NY, USA, 2Department of Biochemistry and Molecular Biology, KUMC, Kansas City, KS, USA, 3Department of Biology, University of Maryland, College Park, MD, USA. GsMTx4 is a spider venom derived peptide selective for cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We created six lysine to glutamate mutants and tested them against Piezo1 channels in outside-out patches and measured lipid binding using a variety of physicochemical assays. Most mutant peptides had lower efficacy (0-40%) compared to wild type (WT). Increased association rates for some of the mutants did not correlate with increased inhibition. And tryptophan fluorescence quenching and isothermal titration calorimetry showed that the free energy of binding to lipid vesicles was largely unaffected by the mutations, suggesting channel inhibition was not a simple function of peptide-lipid association strength. Fluorescence quenching assays showed that WT and mutant peptides bound near the lipid-water interface, but the mutants penetrated deeper than WT. Peptide association as a function of lipid tension was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure (indicated by a shallow slope of the pressure-area isotherms). Mutants with compromised efficacy had steeper slopes in this region suggesting tighter association. These data place GsMTx4 at the membrane surface where it is stabilized by the lysines, and occupying a small fraction of membrane area at normal tension. As surface pressure decreases and free volume increases during membrane stretching, available peptides penetrate deeper acting as outer monolayer ‘‘area reservoirs’’, transferring tension to the inner monolayer. 592-Pos Board B372 The N-Terminal Helix Acts as a Dynamic Membrane Coupler in the Gating Cycle of the Mechanosensitive Channel MscL Navid Bavi1,2, Charles D. Cox1, Paul R. Rohde1, Adam P. Hill1,2, Ben Corry3, Boris Martinac1,2. 1 Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia, 2St Vincent’s Clinical School, Faculty of Medicine, The University of New South Wales, Sydney, Australia, 3 Research School of Biology, The Australian National University, Canberra, Australia. The bacterial mechanosensitive channel of large conductance (MscL) gates in response to membrane tension as a result of force transmitted directly from the lipid bilayer. This channel represents an excellent model system to study the basic principles of mechanosensory transduction. However, despite extensive studies of this homopentameric channel, there is an incomplete understanding of the essential structural components that transduce bilayer tension into channel gating. We postulate that the amphipathic N-terminal helix, which is linked to the pore-lining TM1 helix, acts as a membrane-coupling element that transmits force from the bilayer to the channel pore. To examine this hypothesis we used a multi-scale computational approach, including all-atom molecular dynamics (MD) simulation and finite element (FE) modelling, supported by patch-clamp electrophysiology. Our simulations suggest a strong interaction between the five N-terminal helices of MscL and the lipid bilayer. Moreover, removal of the N-terminal helix, or extending the glycine linker with further (flexible) glycines between the N-terminus and the TM1 helix restricted opening of the channel pore by more than 50%. These computational results were supported by patch-clamp recordings of MscL channels in liposomes and E. coli spheroplasts. Extension of the glycine linker between the N-termini and TM1 helices increased the activation threshold of MscL and resulted in channels that almost exclusively gated in substates. Based on our MD results this is a due to greater conformational freedom of the TM1 helix. These results show that the N-terminus acts as a membrane-coupling element in the
gating cycle of MscL. We suggest this element may represent a common recognisable structure amongst mechanosensitive channels coupling channel conformation to membrane strain. 593-Pos Board B373 Assessing CLC-2 Chloride Channel Voltage Gating by Pore Occupation with Acetate Jose´ J. De Jesu´s-Pe´rez, Jorge Arreola. Institute of Physics, Univ. Autonoma de San Luis Potosi, San Luis Potosi, Mexico. The activation of the homodimeric ClC-2 Cl- channel is voltage dependent. Protopore gates control each of the two pores of the ClC-2 channel. The closed to open transition is initiated by dislodging the negatively charged side chain of a glutamate residue, which constitute the protopore gate located inside the pore. We hypothesize that this gating transition is triggered when intracellular anions occupy the pore. Thus, we investigated channel activation using acetate, a low permeant anion (Pace/PCl = 0.04) that does not traverse the pore. The cells were bathed in a solution containing 140 mM Cl- (pH 7.3) and channel activation was evaluated by measuring the tail current at þ80 mV after application of test pulses from þ40 to 200 mV. When cells were dialyzed with 140 mM acetate there was no activation, perhaps because acetate failed to occupy the pore. To verify this we compared the Vm-dependent activation of ClC-2 in cells dialyzed with acetate þ Cl- (80 þ 60 mM) versus Cl- alone (60 mM). These experiments showed that channel activation was shifted by þ60 mV in cells dialyzed with the acetate þ Cl- mixture relative to that obtained with 60 mM Cl-. Next the charge of the acetate was neutralized by lowering the pHi from 7.3 to 4.2. Since this manipulation ablated the positive shift in activation observed with the mixture we concluded that activation was mediated by multi-ion occupancy. Our data were reproduced by a pore model containing two anion binding sites. Thus, we propose that after a hyperpolarization the intracellular anions occupy the pore and force the gate open to allow anion permeation. Supported by grant 219949 from CONACyT. 594-Pos Board B374 Spontaneous Openings of CFTR are Coupled to Dimer Formation of its Nucleotide Binding Domains Csaba Miha´lyi, La´szlo´ Csana´dy. Dept. of Medical Biochemistry, Semmelweis University, Budapest, Hungary. CFTR pore opening is coupled to dimer formation of its ATP bound nucleotide binding domains (NBDs) and channel closing to disruption of this dimer following the hydrolysis of one of the nucleotides. In addition to such ATP driven openings CFTR infrequently opens also in the absence of any nucleotides. The molecular mechanism behind this latter ‘‘spontaneous’’ activity is unknown because its open probability is far lower than required for reliable kinetic analysis. Previous work showed that transmembrane domain mutations P355A (1) and K978C (2) significantly and additively increase the spontaneous open probability of CFTR. Here we studied spontaneous gating of P355A/K978C CFTR, which is amenable to kinetic analysis, and constructed thermodynamic mutant cycles to test whether such pore openings are coupled to NBD dimer formation. For this purpose, we examined gating-related changes in energetic coupling in our background construct between residues R555 and T1246 on opposing sides of the NBD dimer interface, employing the same two mutations (R555K and T1246N) that were used in the past to demonstrate coupling of pore opening to NBD dimer formation in the presence of ATP (3). We found similar open probabilities and opening rates for background and double mutant (R555K/T1246N) channels, whereas these parameters were significantly reduced for the two single mutants (R555K and T1246N). In contrast, all four constructs displayed similar closing rates. Thus, CFTR residues R555 and T1246 become energetically coupled in the open state (DDGopen-closed=-2.1650.58 kT), and this interaction is already present in the transition state for opening (DDGopening=-2.1050.56 kT; DDGclosing=0.4550.36 kT). Our results suggest that even ATP-free spontaneous pore openings of CFTR are coupled to dimerization of its NBDs. (1) J Biol Chem. 289:19942-19957. (2) PNAS 107:3888-3893. (3) Nature 433:876-880. 595-Pos Board B375 The Prostacyclin Analog Treprostinil Inhibits Ano1-Encoded Ca2D-Activated Cl Channels and Mouse Pulmonary Arterial Tone Through Stimulation of cAMP-Dependent Signaling Pathway Fiona Cunningham, John Lilly, Normand Leblanc. Pharmacology/MS 573, University of Nevada, Reno, Reno, NV, USA.
Sunday, February 28, 2016
into account for screening molecules with improved permeation properties in rational drug designing. 1. Nikaido, H. Microbiol Mol Biol Rev 67, 593-656 (2003). 2. Lou, H. et al. PLoS ONE 6, e25825 (2011). 3. A. Kumar et al. J Phys Chem B 114, 9608-9616 (2010). 4. S. Acosta-Gutierrez. J Phys Chem Lett 6, 1807-1812 (2015). 591-Pos Board B371 GsMTx4 Mutagenesis Reveals Dynamic Membrane Binding Properties that Confer Inhibition of Piezo1 Radhakrishnan Gnanasambandam1, Chiranjib Ghatak2, Anthony Yasmann3, Frederick Sachs1, Alexey S. Ladokhin2, Sergei I. Sukharev3, Thomas M. Suchyna1. 1 Department of Physiology and Biophysics, SUNY University at Buffalo, Buffalo, NY, USA, 2Department of Biochemistry and Molecular Biology, KUMC, Kansas City, KS, USA, 3Department of Biology, University of Maryland, College Park, MD, USA. GsMTx4 is a spider venom derived peptide selective for cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We created six lysine to glutamate mutants and tested them against Piezo1 channels in outside-out patches and measured lipid binding using a variety of physicochemical assays. Most mutant peptides had lower efficacy (0-40%) compared to wild type (WT). Increased association rates for some of the mutants did not correlate with increased inhibition. And tryptophan fluorescence quenching and isothermal titration calorimetry showed that the free energy of binding to lipid vesicles was largely unaffected by the mutations, suggesting channel inhibition was not a simple function of peptide-lipid association strength. Fluorescence quenching assays showed that WT and mutant peptides bound near the lipid-water interface, but the mutants penetrated deeper than WT. Peptide association as a function of lipid tension was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure (indicated by a shallow slope of the pressure-area isotherms). Mutants with compromised efficacy had steeper slopes in this region suggesting tighter association. These data place GsMTx4 at the membrane surface where it is stabilized by the lysines, and occupying a small fraction of membrane area at normal tension. As surface pressure decreases and free volume increases during membrane stretching, available peptides penetrate deeper acting as outer monolayer ‘‘area reservoirs’’, transferring tension to the inner monolayer. 592-Pos Board B372 The N-Terminal Helix Acts as a Dynamic Membrane Coupler in the Gating Cycle of the Mechanosensitive Channel MscL Navid Bavi1,2, Charles D. Cox1, Paul R. Rohde1, Adam P. Hill1,2, Ben Corry3, Boris Martinac1,2. 1 Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, Australia, 2St Vincent’s Clinical School, Faculty of Medicine, The University of New South Wales, Sydney, Australia, 3 Research School of Biology, The Australian National University, Canberra, Australia. The bacterial mechanosensitive channel of large conductance (MscL) gates in response to membrane tension as a result of force transmitted directly from the lipid bilayer. This channel represents an excellent model system to study the basic principles of mechanosensory transduction. However, despite extensive studies of this homopentameric channel, there is an incomplete understanding of the essential structural components that transduce bilayer tension into channel gating. We postulate that the amphipathic N-terminal helix, which is linked to the pore-lining TM1 helix, acts as a membrane-coupling element that transmits force from the bilayer to the channel pore. To examine this hypothesis we used a multi-scale computational approach, including all-atom molecular dynamics (MD) simulation and finite element (FE) modelling, supported by patch-clamp electrophysiology. Our simulations suggest a strong interaction between the five N-terminal helices of MscL and the lipid bilayer. Moreover, removal of the N-terminal helix, or extending the glycine linker with further (flexible) glycines between the N-terminus and the TM1 helix restricted opening of the channel pore by more than 50%. These computational results were supported by patch-clamp recordings of MscL channels in liposomes and E. coli spheroplasts. Extension of the glycine linker between the N-termini and TM1 helices increased the activation threshold of MscL and resulted in channels that almost exclusively gated in substates. Based on our MD results this is a due to greater conformational freedom of the TM1 helix. These results show that the N-terminus acts as a membrane-coupling element in the
gating cycle of MscL. We suggest this element may represent a common recognisable structure amongst mechanosensitive channels coupling channel conformation to membrane strain. 593-Pos Board B373 Assessing CLC-2 Chloride Channel Voltage Gating by Pore Occupation with Acetate Jose´ J. De Jesu´s-Pe´rez, Jorge Arreola. Institute of Physics, Univ. Autonoma de San Luis Potosi, San Luis Potosi, Mexico. The activation of the homodimeric ClC-2 Cl- channel is voltage dependent. Protopore gates control each of the two pores of the ClC-2 channel. The closed to open transition is initiated by dislodging the negatively charged side chain of a glutamate residue, which constitute the protopore gate located inside the pore. We hypothesize that this gating transition is triggered when intracellular anions occupy the pore. Thus, we investigated channel activation using acetate, a low permeant anion (Pace/PCl = 0.04) that does not traverse the pore. The cells were bathed in a solution containing 140 mM Cl- (pH 7.3) and channel activation was evaluated by measuring the tail current at þ80 mV after application of test pulses from þ40 to 200 mV. When cells were dialyzed with 140 mM acetate there was no activation, perhaps because acetate failed to occupy the pore. To verify this we compared the Vm-dependent activation of ClC-2 in cells dialyzed with acetate þ Cl- (80 þ 60 mM) versus Cl- alone (60 mM). These experiments showed that channel activation was shifted by þ60 mV in cells dialyzed with the acetate þ Cl- mixture relative to that obtained with 60 mM Cl-. Next the charge of the acetate was neutralized by lowering the pHi from 7.3 to 4.2. Since this manipulation ablated the positive shift in activation observed with the mixture we concluded that activation was mediated by multi-ion occupancy. Our data were reproduced by a pore model containing two anion binding sites. Thus, we propose that after a hyperpolarization the intracellular anions occupy the pore and force the gate open to allow anion permeation. Supported by grant 219949 from CONACyT. 594-Pos Board B374 Spontaneous Openings of CFTR are Coupled to Dimer Formation of its Nucleotide Binding Domains Csaba Miha´lyi, La´szlo´ Csana´dy. Dept. of Medical Biochemistry, Semmelweis University, Budapest, Hungary. CFTR pore opening is coupled to dimer formation of its ATP bound nucleotide binding domains (NBDs) and channel closing to disruption of this dimer following the hydrolysis of one of the nucleotides. In addition to such ATP driven openings CFTR infrequently opens also in the absence of any nucleotides. The molecular mechanism behind this latter ‘‘spontaneous’’ activity is unknown because its open probability is far lower than required for reliable kinetic analysis. Previous work showed that transmembrane domain mutations P355A (1) and K978C (2) significantly and additively increase the spontaneous open probability of CFTR. Here we studied spontaneous gating of P355A/K978C CFTR, which is amenable to kinetic analysis, and constructed thermodynamic mutant cycles to test whether such pore openings are coupled to NBD dimer formation. For this purpose, we examined gating-related changes in energetic coupling in our background construct between residues R555 and T1246 on opposing sides of the NBD dimer interface, employing the same two mutations (R555K and T1246N) that were used in the past to demonstrate coupling of pore opening to NBD dimer formation in the presence of ATP (3). We found similar open probabilities and opening rates for background and double mutant (R555K/T1246N) channels, whereas these parameters were significantly reduced for the two single mutants (R555K and T1246N). In contrast, all four constructs displayed similar closing rates. Thus, CFTR residues R555 and T1246 become energetically coupled in the open state (DDGopen-closed=-2.1650.58 kT), and this interaction is already present in the transition state for opening (DDGopening=-2.1050.56 kT; DDGclosing=0.4550.36 kT). Our results suggest that even ATP-free spontaneous pore openings of CFTR are coupled to dimerization of its NBDs. (1) J Biol Chem. 289:19942-19957. (2) PNAS 107:3888-3893. (3) Nature 433:876-880. 595-Pos Board B375 The Prostacyclin Analog Treprostinil Inhibits Ano1-Encoded Ca2D-Activated Cl Channels and Mouse Pulmonary Arterial Tone Through Stimulation of cAMP-Dependent Signaling Pathway Fiona Cunningham, John Lilly, Normand Leblanc. Pharmacology/MS 573, University of Nevada, Reno, Reno, NV, USA.