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Mutagenesis Study of the Ca2+ Sensitivity of SK2 Channels
422a
Tuesday, February 14, 2017
as revealed by non-stationary noise analysis. In both activations, the current reverses close to the Nernst potential of Cl-, suggesting that TMEM16A remains highly anion-selective during both activations. We found that the functional behaviour of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. Concatemeric constructs that combine a WT subunit with a subunit with decreased potency exhibit activation properties that can be described by a linear combination of the activation profiles of their constituents. Consistent with this observation, mutation of a putative pore-lining residue that changes the conduction properties of the channel can selectively suppress the activation of the subunit containing this mutation. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca2þ binding to sites that are embedded in the transmembrane part of each subunit. 2074-Pos Board B394 Mutagenesis Study of the Ca2D Sensitivity of SK2 Channels Young Woo Nam, Benjamin J. Whitmore, Razan S. Orfali, Miao Zhang. Chapman University School of Pharmacy, Irvine, CA, USA. Small conductance Ca2þ-activated potassium (SK) channels play a significant role in modulating the membrane excitability of neurons. In animal model studies, SK channels have been identified as a potential drug target for the therapy of movement disorders such as ataxia. Previous studies suggest that the neuronal activity of the Purkinje cells are abnormally elevated in ataxia mouse models. Positive allosteric modulators (PAMs) of SK channels have been shown to increase the Ca2þ sensitivity of these channels, thus reducing the excitability of the cells and exerting neuroprotective effects. The SK channels’ Ca2þ sensitivity is conferred by interaction with calmodulin (CaM), a Ca2þ binding protein. Our previous research has identified the binding pocket of the SK channels’ PAMs to be located between the CaM binding domain (CaMBD) and CaM. These findings have led us to introduce mutations into the PAM binding pocket, resulting in changes in the Ca2þ sensitivity of SK channels. 2075-Pos Board B395 Role of Individual Camp Binding Sites on Relieving the Autoinhibition in HCN Channels Mallikarjuna Rao Sunkara, Jana Kusch, Klaus Benndorf. Institute of Physiology II, University Hospital Jena, Jena, Germany. HCN channels are tetrameric cation channels, activated by hyperpolarizing voltages and modulated by binding of cyclic nucleotides (CN). Each subunit carries an intracellular binding site (CNBD), connected to the transmembrane portion via the C-linker (CL). Several studies proposed that the tetrameric CNBD-CL structure exerts an autoinhibitory effect on the channel, which is relieved by CN binding. The molecular mechanism behind this gating mechanism is still elusive. We want to contribute to a better understanding by studying the contribution of each individual binding step. We monitored HCN2 channel activation following hyperpolarizing voltage jumps in inside-out macro patches with and without saturating [cAMP]. We used concatenated channel constructs with either one, two, three or four disabled binding sites (compare Ulens & Siegelbaum, Neuron, 2003, 40:959-70). We found that occupation of two of the available four CNBDs is sufficient to stabilize the open state. This finding is supported by three types of data: Two ligands were sufficient (1) to reach the maximum current amplitude increase, (2) to slow down depolarization-induced deactivation, and (3) to cause a persistent current during cAMP wash-out. However, to reach maximum open probability, energy provided by voltage had to be higher than at full occupation. In contrast, to evoke the full cAMP-induced shift of the steady-state activation curve to less hyperpolarizing voltages, occupation of all four CNBDs was required. Thereby, each binding site contributed to a similar extent to the shift of the voltage of half-maximum activation (V1/2): one: DV1/2=4.2 mV; two, cis: DV1/2=9.9 mV, two, trans: DV1/2=9.5 mV; three: DV1/2=14.3 mV; four: DV1/2=20.9 mV). Remarkably, both options for half-occupation, cis and trans, led to a similar shift. Together, these data led us conclude that for a complete relief of autoinhibition full occupation of the tetrameric CNBD is required. In partially liganded channels, the relative position of the occupied CNBDs is not important for their effect. 2076-Pos Board B396 Monitoring the Conformational Changes of Individual Cyclic NucleotideGated Ion Channels by High-Speed Atomic Force Microscopy Martina Rangl1, Atsushi Miyagi1, Julia Kowal2, Henning Stahlberg2, Crina M. Nimigean1, Simon Scheuring1. 1 Departments of Anesthesiology, Physiology and Biophysics, and Biochemistry, Weill Cornell Medical College, New York, NY, USA, 2Center
for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland. Eukaryotic cyclic nucleotide-modulated ion channels perform various physiological roles by channel opening in response to cyclic nucleotide binding to a specialized cyclic nucleotide-binding domain (CNBD). Despite increasing knowledge regarding function and structure, a full picture of the gating process is still missing. Here, we report the direct monitoring of ligand-induced conformational changes in single cyclic nucleotide-modulated channels from Mesorhizobium loti (MloK1), in real-time and native-like conditions. Using high-speed atomic force microscopy (HS-AFM) imaging we show that in the presence of cAMP most channels are in a stable conformation characterized by a windmill-like arrangement of the CNBDs within the channel tetramer. A few molecules, however, are observed to dynamically switch back and forth (blink) between states corresponding to at least two CNBD conformations that differ in their height, or distance from the membrane. Upon cAMP depletion more channels start blinking, and the blinking height of an individual channel increases over time suggestive of slow, progressive loss of ligands from each CNBD within the tetramer. Thus, as a fully liganded MloK1 tetramer is exposed to low cAMP, it slowly loses ligands one-by-one from each of the CNBDs, which start blinking independently from each other, yielding a highly mobile state. We propose that during gating, the channel transitions from a mobile set of conformations in the absence of ligand to a stable conformation in the presence of ligand and that these conformations are important for determining the open and closed states of the conducting pore. 2077-Pos Board B397 Identification of an Odorant-Binding Site Residue in an Olfactory Receptor of the Malaria Vector Mosquito Suhaila Rahman, Charles W. Luetje. Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, USA. Many insect behaviors are driven by olfaction, making odorant receptors (ORs) appealing targets for control of disease vector mosquitoes. Insect ORs are odorant-gated ion channels composed of two subunit types; one of many odorant binding subunits and a conserved co-receptor subunit (Orco). We are exploring the structural basis for odorant binding by ORs of the human malaria vector mosquito, Anopheles gambiae. Two structurally and functionally similar odorant binding subunits Agam\Or13 and Agam\Or15 (82% amino acid identity) were expressed in Xenopus oocytes, each in combination with Agam\Orco, and assayed by two-electrode voltage clamp electrophysiology. Both ORs were activated by acetophenone, but displayed distinct sensitivities to antagonism by (-)-fenchone. To identify residue(s) responsible for this difference in antagonist sensitivity, we assayed a series of mutant Agam\Or15 subunits in which each residue that differed between Agam\Or13 and Agam\Or15 in the transmembrane and extracellular regions was changed from the Agam\Or15 residue to the AgamrOr13 residue. Position 195 (Ala in Agam\Or15, Ile in Agam\Or13), located at the interface between the predicted second extracellular loop and fourth transmembrane domain, was found to alter (-)-fenchone sensitivity. The reverse mutation in Agam\Or13 could also alter (-)-fenchone sensitivity. To determine whether residue 195 is located at the odorant binding site of these receptors, we employed thermodynamic mutant cycle analysis. Concentration-inhibition analyses for (-)-fenchone and six structurally related compounds were performed for Agam\Or15 subunits with Ala (wt), Val, Leu and Ile at position 195. High interaction coefficient values obtained for several cycles indicate a close physical proximity between residue 195 and ligand. 2078-Pos Board B398 Modulating O2 Permeability of the Central Pore of RH50 by in Silico Site Directed Mutagenesis Eric Shinn, Emad Tajkhorshid. University of Illinois at Urbana-Champaign, Urbana, IL, USA. The Rhesus (Rh) family of integral membrane proteins exhibit ubiquitous expression among animals and their structural homology to ammonium transport (Amt) proteins suggests their function as ammonia/ammonium transporters. They have also been implicated in facilitating permeation of gaseous species, such as dioxygen and carbon dioxide, across the cell membrane. Rh50 proteins are expressed in erythrocytes, as well as in epithelial tissues of organs such as the kidneys where critical nitrogen processing occurs. Using the solved Rh50 structure from bacterial homologue NeRh50 found in Nitrosomonas europaea, we employed a combination of molecular dynamics and free energy approaches including implicit ligand sampling, explicit ligand sampling, umbrella sampling, and in silico mutagenesis to characterize O2 permeation pathway, mechanism, and energetics. Free-energy calculations reveal high-energy barriers both in the monomeric pore as well as the pore formed
Tuesday, February 14, 2017
as revealed by non-stationary noise analysis. In both activations, the current reverses close to the Nernst potential of Cl-, suggesting that TMEM16A remains highly anion-selective during both activations. We found that the functional behaviour of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. Concatemeric constructs that combine a WT subunit with a subunit with decreased potency exhibit activation properties that can be described by a linear combination of the activation profiles of their constituents. Consistent with this observation, mutation of a putative pore-lining residue that changes the conduction properties of the channel can selectively suppress the activation of the subunit containing this mutation. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca2þ binding to sites that are embedded in the transmembrane part of each subunit. 2074-Pos Board B394 Mutagenesis Study of the Ca2D Sensitivity of SK2 Channels Young Woo Nam, Benjamin J. Whitmore, Razan S. Orfali, Miao Zhang. Chapman University School of Pharmacy, Irvine, CA, USA. Small conductance Ca2þ-activated potassium (SK) channels play a significant role in modulating the membrane excitability of neurons. In animal model studies, SK channels have been identified as a potential drug target for the therapy of movement disorders such as ataxia. Previous studies suggest that the neuronal activity of the Purkinje cells are abnormally elevated in ataxia mouse models. Positive allosteric modulators (PAMs) of SK channels have been shown to increase the Ca2þ sensitivity of these channels, thus reducing the excitability of the cells and exerting neuroprotective effects. The SK channels’ Ca2þ sensitivity is conferred by interaction with calmodulin (CaM), a Ca2þ binding protein. Our previous research has identified the binding pocket of the SK channels’ PAMs to be located between the CaM binding domain (CaMBD) and CaM. These findings have led us to introduce mutations into the PAM binding pocket, resulting in changes in the Ca2þ sensitivity of SK channels. 2075-Pos Board B395 Role of Individual Camp Binding Sites on Relieving the Autoinhibition in HCN Channels Mallikarjuna Rao Sunkara, Jana Kusch, Klaus Benndorf. Institute of Physiology II, University Hospital Jena, Jena, Germany. HCN channels are tetrameric cation channels, activated by hyperpolarizing voltages and modulated by binding of cyclic nucleotides (CN). Each subunit carries an intracellular binding site (CNBD), connected to the transmembrane portion via the C-linker (CL). Several studies proposed that the tetrameric CNBD-CL structure exerts an autoinhibitory effect on the channel, which is relieved by CN binding. The molecular mechanism behind this gating mechanism is still elusive. We want to contribute to a better understanding by studying the contribution of each individual binding step. We monitored HCN2 channel activation following hyperpolarizing voltage jumps in inside-out macro patches with and without saturating [cAMP]. We used concatenated channel constructs with either one, two, three or four disabled binding sites (compare Ulens & Siegelbaum, Neuron, 2003, 40:959-70). We found that occupation of two of the available four CNBDs is sufficient to stabilize the open state. This finding is supported by three types of data: Two ligands were sufficient (1) to reach the maximum current amplitude increase, (2) to slow down depolarization-induced deactivation, and (3) to cause a persistent current during cAMP wash-out. However, to reach maximum open probability, energy provided by voltage had to be higher than at full occupation. In contrast, to evoke the full cAMP-induced shift of the steady-state activation curve to less hyperpolarizing voltages, occupation of all four CNBDs was required. Thereby, each binding site contributed to a similar extent to the shift of the voltage of half-maximum activation (V1/2): one: DV1/2=4.2 mV; two, cis: DV1/2=9.9 mV, two, trans: DV1/2=9.5 mV; three: DV1/2=14.3 mV; four: DV1/2=20.9 mV). Remarkably, both options for half-occupation, cis and trans, led to a similar shift. Together, these data led us conclude that for a complete relief of autoinhibition full occupation of the tetrameric CNBD is required. In partially liganded channels, the relative position of the occupied CNBDs is not important for their effect. 2076-Pos Board B396 Monitoring the Conformational Changes of Individual Cyclic NucleotideGated Ion Channels by High-Speed Atomic Force Microscopy Martina Rangl1, Atsushi Miyagi1, Julia Kowal2, Henning Stahlberg2, Crina M. Nimigean1, Simon Scheuring1. 1 Departments of Anesthesiology, Physiology and Biophysics, and Biochemistry, Weill Cornell Medical College, New York, NY, USA, 2Center
for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland. Eukaryotic cyclic nucleotide-modulated ion channels perform various physiological roles by channel opening in response to cyclic nucleotide binding to a specialized cyclic nucleotide-binding domain (CNBD). Despite increasing knowledge regarding function and structure, a full picture of the gating process is still missing. Here, we report the direct monitoring of ligand-induced conformational changes in single cyclic nucleotide-modulated channels from Mesorhizobium loti (MloK1), in real-time and native-like conditions. Using high-speed atomic force microscopy (HS-AFM) imaging we show that in the presence of cAMP most channels are in a stable conformation characterized by a windmill-like arrangement of the CNBDs within the channel tetramer. A few molecules, however, are observed to dynamically switch back and forth (blink) between states corresponding to at least two CNBD conformations that differ in their height, or distance from the membrane. Upon cAMP depletion more channels start blinking, and the blinking height of an individual channel increases over time suggestive of slow, progressive loss of ligands from each CNBD within the tetramer. Thus, as a fully liganded MloK1 tetramer is exposed to low cAMP, it slowly loses ligands one-by-one from each of the CNBDs, which start blinking independently from each other, yielding a highly mobile state. We propose that during gating, the channel transitions from a mobile set of conformations in the absence of ligand to a stable conformation in the presence of ligand and that these conformations are important for determining the open and closed states of the conducting pore. 2077-Pos Board B397 Identification of an Odorant-Binding Site Residue in an Olfactory Receptor of the Malaria Vector Mosquito Suhaila Rahman, Charles W. Luetje. Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, USA. Many insect behaviors are driven by olfaction, making odorant receptors (ORs) appealing targets for control of disease vector mosquitoes. Insect ORs are odorant-gated ion channels composed of two subunit types; one of many odorant binding subunits and a conserved co-receptor subunit (Orco). We are exploring the structural basis for odorant binding by ORs of the human malaria vector mosquito, Anopheles gambiae. Two structurally and functionally similar odorant binding subunits Agam\Or13 and Agam\Or15 (82% amino acid identity) were expressed in Xenopus oocytes, each in combination with Agam\Orco, and assayed by two-electrode voltage clamp electrophysiology. Both ORs were activated by acetophenone, but displayed distinct sensitivities to antagonism by (-)-fenchone. To identify residue(s) responsible for this difference in antagonist sensitivity, we assayed a series of mutant Agam\Or15 subunits in which each residue that differed between Agam\Or13 and Agam\Or15 in the transmembrane and extracellular regions was changed from the Agam\Or15 residue to the AgamrOr13 residue. Position 195 (Ala in Agam\Or15, Ile in Agam\Or13), located at the interface between the predicted second extracellular loop and fourth transmembrane domain, was found to alter (-)-fenchone sensitivity. The reverse mutation in Agam\Or13 could also alter (-)-fenchone sensitivity. To determine whether residue 195 is located at the odorant binding site of these receptors, we employed thermodynamic mutant cycle analysis. Concentration-inhibition analyses for (-)-fenchone and six structurally related compounds were performed for Agam\Or15 subunits with Ala (wt), Val, Leu and Ile at position 195. High interaction coefficient values obtained for several cycles indicate a close physical proximity between residue 195 and ligand. 2078-Pos Board B398 Modulating O2 Permeability of the Central Pore of RH50 by in Silico Site Directed Mutagenesis Eric Shinn, Emad Tajkhorshid. University of Illinois at Urbana-Champaign, Urbana, IL, USA. The Rhesus (Rh) family of integral membrane proteins exhibit ubiquitous expression among animals and their structural homology to ammonium transport (Amt) proteins suggests their function as ammonia/ammonium transporters. They have also been implicated in facilitating permeation of gaseous species, such as dioxygen and carbon dioxide, across the cell membrane. Rh50 proteins are expressed in erythrocytes, as well as in epithelial tissues of organs such as the kidneys where critical nitrogen processing occurs. Using the solved Rh50 structure from bacterial homologue NeRh50 found in Nitrosomonas europaea, we employed a combination of molecular dynamics and free energy approaches including implicit ligand sampling, explicit ligand sampling, umbrella sampling, and in silico mutagenesis to characterize O2 permeation pathway, mechanism, and energetics. Free-energy calculations reveal high-energy barriers both in the monomeric pore as well as the pore formed