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Molecular Locations of Gates in Potassium Channels
510a
Wednesday, March 2, 2016
Symposium: Voltage Sensing and Gating 2513-Symp Conformational Changes during Voltage Sensing Francisco Bezanilla, Jerome Lacroix, Michael Priest. Dept Biochem/Molec Bio, Univ Chicago, Chicago, IL, USA. Voltage sensing in S4-based voltage sensors operate by translocating charge in the membrane electric field. Typically these charges are four arginines in the S4 helix, each separated by two hydrophobic residues. The movement of the arginines displace the helix which, in turn opens (or closes) the conduction pathway or activates a phosphatase. Although we have detailed information about the magnitude and kinetics of the charge movement and the basic structure of several voltage sensors we still have limited knowledge as of how the actual translocation occurs and how it is modulated by the structure. We use a combination of gating current measurements with fluorescence spectroscopy and site-directed mutagenesis to understand the pathway of the gating charges during voltage sensing. In the major states of the sensor, resting, active and relaxed the arginines reside mostly in an hydrophilic environment but at least one of them must be within the electric field so that when the membrane potential is changed it can feel it to cross the hydrophobic plug. The crossing requires that the arginines make their way through a completely closed pathway, as revealed by the crystal structure. We have investigated the nature of the side chains that form the plug and studied how they regulate the region where the field is located, so that the arginines sense the voltage change, and how they allow the passage of the arginines. Several amino acids are critical for these two functions and at the same time some of them determine the setpoint of activation and the energy barrier that controls the kinetics with clear differences between the fast Na channels and the slow K channels. The control imposed by the plug residues is different for each of the sensing arginines which in turn have different pathways. Support: NIHGM030376. 2514-Symp Voltage-Sensing Domains as Ion Channels Francesco Tombola. Dept Physio/Biophys, Univ California, Irvine, Irvine, CA, USA. The voltage-sensing domain (VSD) of the voltage-gated channel Hv1 is inherently proton-conductive and will be discussed as a paradigm of VSD-mediated ion conduction. Special emphasis will be given to the relationship between voltage-gating and the block of ion conduction by small molecules. 2515-Symp Molecular Locations of Gates in Potassium Channels Crina Nimigean. Anesthesiology, Weill Medical College of Cornell University, New York, NY, USA. Understanding how ion channels open and close their pores is crucial for understanding their physiological roles. We used intracellular quaternary ammonium blockers to locate the voltage and calcium-dependent gates in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology, stopped-flow spectrofluorometry, and X-ray crystallography. Blockers bind in an aqueous cavity between two putative gates, an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location: an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. A kinetic single-channel analysis of tetrabutylammonium block of MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutylantimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggests that MthK gating with voltage also leads to conformational changes within the cavity and intracellular pore entrance. For locating the calcium gate, we employed a Tlþ flux assay using a stoppedflow spectrofluorometer. MthK channel activity was estimated from the rate of a MthK-containing liposome-trapped fluorophore quenching due to Tlþ influx through the channels. The high-affinity blocker tetrapentylammonium, applied prior to channel activation using a sequential mixing protocol, was able to fully block closed channels, indicating that the blocker can reach its binding site in closed channels. Given that the blocker binding site is in the cytoplasmic access below the selectivity filter, these results suggest that there is also a calcium-dependent gate at the selectivity filter in MthK.
2516-Symp Mechanisms of KCNE Beta Subunit Modulation of Voltage Sensing and Gating in KCNQ1 Channels Peter Larsson. University of Miami, Miami, FL, USA. KCNQ1 (Kv7.1) channels are expressed in different tissues and exhibit different gating behaviors in these different tissues. In the heart, KCNQ1 channels are coexpressed with KCNE1 beta subunits and form slowly activating, voltage-gated potassium channels, whereas in epithelial cells KCNQ1 are coexpressed with KCNE2 or KCNE3 beta subunits and form potassium channels that appear voltage-independent. We have used voltage clamp fluorometry (VCF) to elucidate the gating mechanism in KCNQ1 channels and the effects of KCNE beta subunits on KCNQ1 gating. Voltage clamp fluorometry allows us to follow both voltage sensor movement and gate opening simultaneously, thereby better understand the relationship of voltage sensor movement and channel opening in these channels. We show that the voltage sensor S4 moves and the gate closes in KCNQ1/KCNE3 channels, but only at very negative voltages. By decoupling the voltage sensor and the gate, we show that KCNE3 mainly affects the voltage sensor and only indirectly affects the pore through the voltage-sensor-to-gate coupling. The KCNE3 seems to affect the voltage sensor S4 via an electrostatic interactions between negatively charged KCNE3 residues and positively charged S4 residues. Our data suggests that KCNE1 and KCNE3 affect different domains of KCNQ1, thereby resulting in very different modulations of KCNQ1 voltage gating. KCNE1 affects both the voltage-sensing domain and the pore-gate domain, whereas KCNE3 affects mainly the voltage-sensing domain. That different KCNE beta subunits affect different domains of KCNQ1 explains how two highly similar KCNE beta subunits have so different effects on KCNQ1 gating.
Symposium: Chemomechanical Coupling in Immune Response 2517-Symp Signaling Reactions on Membrane Surfaces: The Roles of Space, Force, and Time Jay Groves. Univ California Berkeley, Berkeley, CA, USA. The cell membrane is a two-dimensional fluid emulsion within which a majority of cellular signaling reactions take place. Among the many possible ways physical aspects of the membrane surface can impact these signaling processes, lipid immiscibility and the tendency towards demixing as well as the restriction of orientational degrees of freedom stand out as especially significant. I will describe results from several newly reconstituted membrane signaling systems that highlight this significant and sometimes surprising impact of membranes on the chemistry of cellular signal transduction. 2518-Symp Chemomechanical Interactions Enhance IgE-FcεRI Signaling in Mast Cells Barbara Baird. Chemistry, Cornell Univ, Ithaca, NY, USA. Cells respond to their physical environment and to chemical stimuli in terms of collective molecular interactions that are regulated in time and space. Small molecules may engage specific receptors to initiate a transmembrane signal, and the system amplifies this nanoscale interaction to microscale assemblies within the cell and often to longer length scales involving surrounding tissue and ultimately the whole organism. A striking example of signal integration over multiple length scales is the allergic immune response. IgE receptors (FcεRI) on mast cells are the gatekeepers of this response, and this system has proven to be a valuable model for investigating receptor-mediated cellular activation. Spanning the range of cellular responses we use super resolution fluorescence localization microscopy to investigate the earliest signaling events and ligands patterned in micron size features together with confocal microscopy to investigate early and later events. We are characterizing distinctive regulatory roles played by the actin cytoskeleton at different stages. We are further investigating spatial redistributions of the cellular integrins that relate to the enhancement of signaling observed for cells spreading on surfaces. 2519-Symp Protein Nanoclustering as Funcional Unit of Immune Cells Maria Garcia-Parajo PhD1,2. 1 ICFO-Institute of Photonic Sciences, Castelldefels, Barcelona, Spain, 2 ICREA-Institucio´ Catalana de Recerca i Estudis Avanc¸ats, Barcelona, Spain.
Wednesday, March 2, 2016
Symposium: Voltage Sensing and Gating 2513-Symp Conformational Changes during Voltage Sensing Francisco Bezanilla, Jerome Lacroix, Michael Priest. Dept Biochem/Molec Bio, Univ Chicago, Chicago, IL, USA. Voltage sensing in S4-based voltage sensors operate by translocating charge in the membrane electric field. Typically these charges are four arginines in the S4 helix, each separated by two hydrophobic residues. The movement of the arginines displace the helix which, in turn opens (or closes) the conduction pathway or activates a phosphatase. Although we have detailed information about the magnitude and kinetics of the charge movement and the basic structure of several voltage sensors we still have limited knowledge as of how the actual translocation occurs and how it is modulated by the structure. We use a combination of gating current measurements with fluorescence spectroscopy and site-directed mutagenesis to understand the pathway of the gating charges during voltage sensing. In the major states of the sensor, resting, active and relaxed the arginines reside mostly in an hydrophilic environment but at least one of them must be within the electric field so that when the membrane potential is changed it can feel it to cross the hydrophobic plug. The crossing requires that the arginines make their way through a completely closed pathway, as revealed by the crystal structure. We have investigated the nature of the side chains that form the plug and studied how they regulate the region where the field is located, so that the arginines sense the voltage change, and how they allow the passage of the arginines. Several amino acids are critical for these two functions and at the same time some of them determine the setpoint of activation and the energy barrier that controls the kinetics with clear differences between the fast Na channels and the slow K channels. The control imposed by the plug residues is different for each of the sensing arginines which in turn have different pathways. Support: NIHGM030376. 2514-Symp Voltage-Sensing Domains as Ion Channels Francesco Tombola. Dept Physio/Biophys, Univ California, Irvine, Irvine, CA, USA. The voltage-sensing domain (VSD) of the voltage-gated channel Hv1 is inherently proton-conductive and will be discussed as a paradigm of VSD-mediated ion conduction. Special emphasis will be given to the relationship between voltage-gating and the block of ion conduction by small molecules. 2515-Symp Molecular Locations of Gates in Potassium Channels Crina Nimigean. Anesthesiology, Weill Medical College of Cornell University, New York, NY, USA. Understanding how ion channels open and close their pores is crucial for understanding their physiological roles. We used intracellular quaternary ammonium blockers to locate the voltage and calcium-dependent gates in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology, stopped-flow spectrofluorometry, and X-ray crystallography. Blockers bind in an aqueous cavity between two putative gates, an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location: an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. A kinetic single-channel analysis of tetrabutylammonium block of MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutylantimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggests that MthK gating with voltage also leads to conformational changes within the cavity and intracellular pore entrance. For locating the calcium gate, we employed a Tlþ flux assay using a stoppedflow spectrofluorometer. MthK channel activity was estimated from the rate of a MthK-containing liposome-trapped fluorophore quenching due to Tlþ influx through the channels. The high-affinity blocker tetrapentylammonium, applied prior to channel activation using a sequential mixing protocol, was able to fully block closed channels, indicating that the blocker can reach its binding site in closed channels. Given that the blocker binding site is in the cytoplasmic access below the selectivity filter, these results suggest that there is also a calcium-dependent gate at the selectivity filter in MthK.
2516-Symp Mechanisms of KCNE Beta Subunit Modulation of Voltage Sensing and Gating in KCNQ1 Channels Peter Larsson. University of Miami, Miami, FL, USA. KCNQ1 (Kv7.1) channels are expressed in different tissues and exhibit different gating behaviors in these different tissues. In the heart, KCNQ1 channels are coexpressed with KCNE1 beta subunits and form slowly activating, voltage-gated potassium channels, whereas in epithelial cells KCNQ1 are coexpressed with KCNE2 or KCNE3 beta subunits and form potassium channels that appear voltage-independent. We have used voltage clamp fluorometry (VCF) to elucidate the gating mechanism in KCNQ1 channels and the effects of KCNE beta subunits on KCNQ1 gating. Voltage clamp fluorometry allows us to follow both voltage sensor movement and gate opening simultaneously, thereby better understand the relationship of voltage sensor movement and channel opening in these channels. We show that the voltage sensor S4 moves and the gate closes in KCNQ1/KCNE3 channels, but only at very negative voltages. By decoupling the voltage sensor and the gate, we show that KCNE3 mainly affects the voltage sensor and only indirectly affects the pore through the voltage-sensor-to-gate coupling. The KCNE3 seems to affect the voltage sensor S4 via an electrostatic interactions between negatively charged KCNE3 residues and positively charged S4 residues. Our data suggests that KCNE1 and KCNE3 affect different domains of KCNQ1, thereby resulting in very different modulations of KCNQ1 voltage gating. KCNE1 affects both the voltage-sensing domain and the pore-gate domain, whereas KCNE3 affects mainly the voltage-sensing domain. That different KCNE beta subunits affect different domains of KCNQ1 explains how two highly similar KCNE beta subunits have so different effects on KCNQ1 gating.
Symposium: Chemomechanical Coupling in Immune Response 2517-Symp Signaling Reactions on Membrane Surfaces: The Roles of Space, Force, and Time Jay Groves. Univ California Berkeley, Berkeley, CA, USA. The cell membrane is a two-dimensional fluid emulsion within which a majority of cellular signaling reactions take place. Among the many possible ways physical aspects of the membrane surface can impact these signaling processes, lipid immiscibility and the tendency towards demixing as well as the restriction of orientational degrees of freedom stand out as especially significant. I will describe results from several newly reconstituted membrane signaling systems that highlight this significant and sometimes surprising impact of membranes on the chemistry of cellular signal transduction. 2518-Symp Chemomechanical Interactions Enhance IgE-FcεRI Signaling in Mast Cells Barbara Baird. Chemistry, Cornell Univ, Ithaca, NY, USA. Cells respond to their physical environment and to chemical stimuli in terms of collective molecular interactions that are regulated in time and space. Small molecules may engage specific receptors to initiate a transmembrane signal, and the system amplifies this nanoscale interaction to microscale assemblies within the cell and often to longer length scales involving surrounding tissue and ultimately the whole organism. A striking example of signal integration over multiple length scales is the allergic immune response. IgE receptors (FcεRI) on mast cells are the gatekeepers of this response, and this system has proven to be a valuable model for investigating receptor-mediated cellular activation. Spanning the range of cellular responses we use super resolution fluorescence localization microscopy to investigate the earliest signaling events and ligands patterned in micron size features together with confocal microscopy to investigate early and later events. We are characterizing distinctive regulatory roles played by the actin cytoskeleton at different stages. We are further investigating spatial redistributions of the cellular integrins that relate to the enhancement of signaling observed for cells spreading on surfaces. 2519-Symp Protein Nanoclustering as Funcional Unit of Immune Cells Maria Garcia-Parajo PhD1,2. 1 ICFO-Institute of Photonic Sciences, Castelldefels, Barcelona, Spain, 2 ICREA-Institucio´ Catalana de Recerca i Estudis Avanc¸ats, Barcelona, Spain.