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Shaker-IR K Channel Gating in Heavy Water: Role of Structural Water Molecules in Inactivation
Tuesday, March 1, 2016 nanodomains can form and exhibit coupling between inner and outer leaflets ordered domains. Methods to prepare a wider range of asymmetric membrane compositions and make more facile asymmetry measurements are being developed. In addition, cyclodextrin-catalyzed lipid exchange is being extended to studies of membrane domain structure and function in living cells. Substituting for cholesterol shows the subset of sterols having the ability to support the formation of lipid rafts are necessary and sufficient for them to support membrane domain formation in Borrelia burgdorferi, a bacteriumcontaining fatty acyl cholesterol glycosides.Interestingly,we find fatty acyl cholesterol glycosides are able to form ordered membrane domains without sphingolipids. Studies with cyclodextrin-catalyzed sterol exchange in mammalian cells show that sterols having an ability to promote the formation of ordered membrane domains are necessary and sufficient for them to support both clathrin-dependent and independent endocytosis provided they also contain a 3-beta OH. Efforts to carry out similar experiments with phospholipid and sphingolipid exchange are underway. 1691-Symp The Biophysics of Living Membranes: Protein Partitioning and Functional Differentiation in Ordered Plasma Membrane Domains Ilya Levental. Integrative Biology and Pharmacology, University of Texas Medical School at Houston, Houston, TX, USA. The lipid raft hypothesis posits that ordered, lateral membrane domains are important for the regulation of plasma membrane (PM) structure and function in eukaryotes. Giant Plasma Membrane Vesicles (GPMVs) are isolated plasma membranes that microscopically separate into coexisting ordered and disordered phases, facilitating experimental analysis of the composition and physical properties of ordered raft domains in biological membranes. In these studies, we analyze the biophysical and lipidomic differentiation of the plasma membrane in Mesenchymal Stem Cells (MSC) as the cells undergo differentiation into adipocytes and osteoblasts. During differentiation, dramatic remodeling the PM lipidome, including lipid acyl chain length and unsaturation, results inchanges to membrane order and phase separation. These observations elucidate the compositional determinants of biophysical properties in biological membranes, as well as identify lineage-specific PM features. These results facilitate rational remodeling of membrane phenotypes to direct differentiation, as supplementation with -3 docosahexaneoic acid (DHA) promoted the osteoblastic PM phenotype and potentiated osteogenic differentiation. The differences in the physical properties of the coexisting domains lead to preferential protein partitioning between them. We evaluate the structural determinants of raft partitioning of a model transmembrane protein, and find that raft phase partitioning is related to features of the protein’s transmembrane domain (TMD), namely the hydrophobic length and surface area of the TMD. Longer TMDs impart greater raft association while TMDs with larger, bulkier amino acid side chains prefer the non-raft phase. We present a simple physical model wherein raft partitioning is driven by phase-dependent differences in interfacial energy between the TMD and its surrounding lipid matrix, and find excellent quantitative agreement with observations that provide the first predictions of protein-lipid surface tension. These results point the way to a general rule for raft partitioning of transmembrane proteins.
Platform: Voltage-gated K Channels, Mechanisms of Voltage Sensing and Gating II 1692-Plat Molecular Dynamics Simulations of Hydrophobic Matching in KcsA Karen M. Callahan1, Benoit Mondou2, Louis Sasseville1, Jean-Louis Schwartz1, Jurgen Sygusch2, Nazzareno D’Avanzo1. 1 De´partment de Physiologie Mole´culaire et Inte´grative, Universite´ de Montre´al, Montre´al, QC, Canada, 2De´partment de Biochimie et Me´decine Mole´culaire, Universite´ de Montre´al, Montre´al, QC, Canada. Ion channels are regulated by many factors, including their interactions with lipids. While some lipid-protein interactions may be specific, acting as a ligand, membranes can also influence protein activity through their physiochemical properties, including hydrophobic thickness. Certain conformations of proteins can be preferentially stabilized or destabilized in a membrane-dependent fashion through matching of hydrophobic and hydrophilic portions of the surfaces of the protein and the membrane. Here, we examine atomic-scale molecular dynamics simulations of WT and E71A KcsA in heterogenous membranes of phosphatidylcholine and phosphatidylglycerol lipids of differing thickness supported by our experimental studies of electrophysiological activity and small angle X-ray scattering (SAXS).
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1693-Plat Insights into Ion Channel Selectivity with Ionic Coulomb Blockade William A.T. Gibby1, Dmitri G. Luchinsky2,3, Igor Kh Kaufman2, Peter V.E. McClintock2, Aneta Stefanovska2, Robert S. Eisenberg4. 1 University of Lancaster, Lancaster, United Kingdom, 2Department of Physics, University of Lancaster, Lancaster, United Kingdom, 3Mission Critical Technologies, El Segundo, CA, USA, 4Department of Molecular Biophysics, Rush University Medical Center, Chicago, IL, USA. The flow of ions through a biological ion channel can be considered as transitions between occupied energy levels in the channel and either of the connecting bulk reservoirs [2]. Discreteness of ions and an electrostatic exclusion principle ensure that the number of channel energy levels equals the number of occupying ions. Using these fundamental physical principles we have recently introduced [1] an ionic Coulomb blockade (ICB) theory developed by analogy with the similar phenomenon of electron tunnelling in quantum dots [2,3]. In this picture channel selectivity is governed by energy level changes [1]. We present details of the ICB theory for ion transitions through the channel. It incorporates physiological solutions and channel properties: physical dimension, voltage drop and fixed charge, and hence allows for comparison with physiological data. The set of kinetic equations obtained using ICB is analysed. The channel probability of occupancy as a function of transition rates (and hence fixed charge and number of ions) is obtained in the steady-state approximation. It is shown that this probability displays the staircase structure familiar from analysis of occupancy in quantum dots. It is also shown that current through the channel displays sharp peaks as a function of fixed charge, hence relating channel selectivity to the structure and position of energy levels. The contribution of hydration energy is also discussed. We anticipate that inclusion of this energy into ICB theory will provide an important insight into the selectivity and conductivity of ion channels. [1] Kaufman, I. K., McClintock, P. V. E., Eisenberg, R. S., New Journal of Physics. 17, 8, 15 (2015). [2] Beenakker, C.W.J., Phys. Rev. B., 44, 4, (1991). [3] Krems, M., Di Ventra, M., Phys. Condens. Matter, 25, 6, 065101 (2013). 1694-Plat Ionic Basis of Repolarization of Atrial and Ventricular Specific Cell Types Derived from Human Induced Pluripotent Stem Cells Aaron D. Kaplan1, Randall L. Rasmusson1, Glenna C.L. Bett2. 1 Physiology and Biophysics, Buffalo, NY, USA, 2OB/Gyn, Buffalo, NY, USA. Cardiomyocytes derived from human induced pluripotent stem cells (hIPSCCMs) are an innovative cellular system for understanding human cardiac pathology and physiology. However, hIPSC-CMs express low levels of inward rectifying potassium channel (IK1) relative to native cardiac cells. The lack of this current deforms the action potential (AP) and leads to a depolarized or spontaneous diastolic potential. We used electronic expression of IK1 via dynamic clamp to restore the resting membrane potential back to physiological levels. This resulted in an improved AP morphology, including a reduction in variability and a rate dependent spike and dome shape. There were several significant differences between atrial and ventricular cells including differences in cell capacitance, sodium current magnitude and kinetics and most prominently differences in the transient outward currents. The late components of outward current, cells with atrial APs had a significantly larger sustained outward IKUR or Kv1.5-like component at þ50 mV than ventricular shaped APs (in pA/pF: 3.71 5 0.55 (n=5) vs 1.00 5 0.10 (n=16),P<0.05) but similar peak outward currents: (6.89 5 0.50 (n=5) vs. 6.58 5 0 .67 (n=14),P=N.S). This plateau current is strongly inhibited by 50 micro molar 4-aminopyridine(4-AP). Similarly, atrial-like APs took on a ventricular like shape when treated with 4-AP while the ventricular myocytes APs were 4-AP insensitive. A cloned Kv1.5 current was expressed in oocytes and was used through the electronic expression system to add an IKur to the ventricular myocytes. Addition of this current changed the action potential morphology from ventricle to atrial like. This strongly suggests that IKur is the major determinant of atrial action potential morphology. 1695-Plat Shaker-IR K Channel Gating in Heavy Water: Role of Structural Water Molecules in Inactivation Tibor G. Szanto1, Szabolcs M. Gaal1, Zoltan Varga2, Gyorgy Panyi1. 1 Dept. of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary, 2MTA-DE-NAP B Ion Channel Structure-Function Research Group, University of Debrecen, Debrecen, Hungary. It has been reported earlier that the slow (C-type) inactivated conformation in eukaryotic KV channels is stabilized by a multipoint hydrogen-bond network
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behind the selectivity filter. Furthermore, the selectivity filter is sterically locked in the inactive conformation by buried water molecules and the binding of these ‘hidden’ molecules influence the inactivation process. We found that applying a heavy water (deuterium oxide, D2O)-based extracellular solution dramatically slowed the entry into the inactivated state which might indicate the role of the ‘‘structural water’’ molecules in the conformational stability of the selectivity filter. Alternatively, these observations can be explained by an increase in the viscosity or an altered residency time/exit rate of Kþ from the selectivity filter in a D2O-based extracellular solution. We mimicked the increased viscosity by adding glycerol to the extracellular solution and determined the inactivation kinetics in Shaker-IR channels having the following mutations and allowing slow inactivation at various rates: T449A and T449A/ I470A. The channels were transiently expressed in tsA_201 cells and ionic current experiments were recorded from either inside-out or outside-out patches. We found that application of 5% glycerol had negligible effect on the rate of inactivation kinetics. The exit rate of Kþ ions was studied by changing the Kþ gradient to allow the inactivation time constants to be determined for both outward and inward currents. Our results showed that exposure of the patches to extracellular D2O did not change the toutward/tinward ratio as compared to control (H2O on both sides). Therefore, our macroscopic current measurements support the hypothesis that structural water molecules may have specific effects on the inactivation kinetics by accessing to the region behind the selectivity filter, as proposed by molecular modelling data. 1696-Plat An Intrinsic Ligand Mediates N- and C-Terminal Interactions with the KCNH Gating Machinery Yaxian Zhao1, Joao H. Morais-Cabral2, Andreia Sousa Fernandes2, Gail A. Robertson1. 1 Department of Neuroscience, UW-Madison, Madison, WI, USA, 2Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal. EAG, ERG and ELK are members of the KCNH potassium channel family important in excitability. Two conserved domains in the cytosolic N and C termini uniquely characterize members of this family. The C-terminal domain has homology to the cyclic nucleotide-binding domain of CNG/HCN channels but is unaffected by cyclic nucleotides. At the tail end of this cyclic nucleotidebinding homology domain (CNBhD) is an intrinsic ligand comprising two highly conserved residues that loop back to occupy the cyclic nucleotidebinding pocket. The CNBhD interacts with the N-terminal domain, with one interface also occupied by the intrinsic ligand. We probed the intrinsic ligand and its effect on C- and N-terminal modulation of gating in hEAG1 channels. Using two-electrode voltage clamp of channels expressed in Xenopus oocytes, we found that substitution of the intrinsic ligand residues, Y699 and L701, with AA, SS and GG progressively shifted the midpoint of the conductance-voltage relationship to more depolarized potentials. The substitution with G, a residue unable to support a ligand-receptor interaction and thus considered fully unliganded, resulted in the greatest steady-state shift of channels into the closed state. Interestingly, deleting the CNBhD created the same phenotype as the GG mutant, suggesting the unliganded conformation confers a loss of function of the CNBhD’s effects on gating. Both AA and GG mutants abolished in vitro binding of the PAS domain to the CNBhD in anisotropy measurements. Functionally, when combining GG/CNBhD deletion with mutations at PAS-CNBhD interfaces, we observed a non-additive phenotype that is indistinguishable from the GG or CNBhD deletion mutant. These findings suggest that both the intrinsic ligand and CNBhD are required for the proper function of PAS domain, and the ligand and binding pocket can adopt multiple conformations, each conferring a different gating behavior. 1697-Plat Targeted Enhancement of hERG KD Channel Activity with scFv Antibody Fragments Greg Starek1, Carol A. Harley2, David K. Jones1, Gail A. Robertson1, Joa˜o H. Morais-Cabral2. 1 University of Wisconsin, Madison, WI, USA, 2IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal. Cardiac IKr is a repolarizing current comprising subunits encoded by the human ether-a-go-go related gene (hERG). A defining feature of hERG and related channel types is an N-terminal PAS domain that, through its effects on gating, reduces current amplitude. To probe the functional consequences of disrupting this natural channel modulation, we screened for single chain fragment variable (scFv) antibodies against the PAS domain using phage display. We assessed the binding properties to PAS and functional consequences of these antibodies on the hERG channel. Two isolates affecting hERG 1a current properties were shown by GST pull-down assays to interact with different regions of the PAS domain with submicromolar affinities. In
HEK-293 cells stably expressing hERG 1a, one antibody slowed inactivation onset and the other accelerated recovery from inactivation. Both significantly increased repolarizing current during a voltage protocol mimicking a ventricular action potential. Because native IKr channels are heteromers of the original hERG 1a isolate and hERG 1b, an isoform from which the PAS domain is transcriptionally omitted, we tested the effect of the scFv antibodies on cardiomyocytes derived from human induced pluripotent stem cells. Similar to the HEK-293 findings, the antibodies diminished channel rectification and significantly increased the repolarizing charge during a ventricular action potential voltage clamp. Here, we demonstrate the versatility of scFv antibodies as a site-specific probe of ion channel function. Given their ability to increase IKr, we propose the PAS-channel interface as a therapeutic target and the use of scFv antibodies as a novel treatment for diseases of excitability where action potential duration is prolonged. 1698-Plat Functional and Crystallographic Studies of Hetero-Multimeric KD Channels Spandana Vemulapally, D. Marien Cortes, Luis G. Cuello. Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Science Center, Lubbock, TX, USA. Voltage gated potassium channels (Kv channels) consist of four identical subunits that form a central permeation pathway in which changes in diameter of an inner constriction known as the activation gate (AG) catalyzes their transition from the closed to the open state. Kv channels play a major role in shaping and regulating the action potential in all excitable cells. Expressed as homotetramers as well as heterotetramers, Kv channels have a crucial role in excitability disorders such as arrhythmias, long QT syndrome, hyperactivity disorders, epilepsia and many other behavioral disorders. This renders heteromeric Kv channels as potential pharmaceutical targets but challenges arise because of their hetero-multimeric nature and diverse sequence identity, which make the smart design of safer and more specific therapeutic drugs difficult. Thus, it is of significant importance to study in detail the structure-function relationship in heteromeric Kv channels. Since KcsA is the bona fide structural and functional surrogate of the pore domain of Kv channels, we have constructed tandem-tetramer constructs of KcsA in which all four protomers are expressed as a single polypeptide chain by engineering linkers between the protomers. We were able to express, purify and solved the crystal structure of functional KcsA tandem-tetramer. This opens up a new and unprecedented avenue to introduce dose dependent mutations in the channel tetramer to evaluate the cooperative behavior of the channel function i.e., ion permeation, gating and ion selectivity, from a functional and structural point of view by using electrophysiology and X-ray crystallography approaches. Founding: AHA-11SDG5440003, NIH 1RO1GMo97159-01A1 and Welch Foundation BI-1757 1699-Plat Optical Recording of Voltage Activation of Endogenous Potassium Channels Mark W. Lillya1, Laxmi K. Parajuli2, Sebastian Fletcher-Taylor1, Joyce Huang1, Bruce E. Cohen3, Karen Zito2, Jon T. Sack1. 1 Physiology and Membrane Biology, University of California, Davis, Davis, CA, USA, 2Center for Neuroscience, University of California, Davis, Davis, CA, USA, 3Biological Nanostructures Facility, Laurence Berkeley National Laboratory, Berkeley, CA, USA. Diverse cellular electrical signals are conducted by a vast array of ion channel types acting in concert. The spatial and temporal contributions of specific voltage gated ion channels to neuronal electrical signaling are challenging to establish. We have developed fluorescent probes that indicate when and where Kv2 voltage gated potassium channels are activated by voltage. These ion channel activity probes are engineered from tarantula toxins that bind Kv2 channel voltage sensors at rest, and dissociate when the channels are voltage activated. Previously, we found ion channel activity probes to detect overexpressed Kv2 ion channels. Here, we explore their ability to identify activation of endogenous neuronal ion channels. In rat hippocampal slices and dissociated neurons, we find that exogenously applied Kv2 ion channel probes label punctate structures on neuronal cell bodies similar to endogenous Kv2 ion channels. In neurons transfected with GFP-Kv2.1, ion channel probe puncta colocalize with GFP puncta. Depolarizing stimuli that induce Kv2 channel activation, trigger dissipation of fluorescent puncta from neurons. Together, our results suggest that ion channel probes can identify the locations of specific ion channel types in living neurons, and report when and where the channels are activated by voltage. Further development of probes that target to select conformations of ion channels could enable imaging of the contributions of specific ion channel types to electrical activity in neuronal networks.
Platform: Voltage-gated K Channels, Mechanisms of Voltage Sensing and Gating II 1692-Plat Molecular Dynamics Simulations of Hydrophobic Matching in KcsA Karen M. Callahan1, Benoit Mondou2, Louis Sasseville1, Jean-Louis Schwartz1, Jurgen Sygusch2, Nazzareno D’Avanzo1. 1 De´partment de Physiologie Mole´culaire et Inte´grative, Universite´ de Montre´al, Montre´al, QC, Canada, 2De´partment de Biochimie et Me´decine Mole´culaire, Universite´ de Montre´al, Montre´al, QC, Canada. Ion channels are regulated by many factors, including their interactions with lipids. While some lipid-protein interactions may be specific, acting as a ligand, membranes can also influence protein activity through their physiochemical properties, including hydrophobic thickness. Certain conformations of proteins can be preferentially stabilized or destabilized in a membrane-dependent fashion through matching of hydrophobic and hydrophilic portions of the surfaces of the protein and the membrane. Here, we examine atomic-scale molecular dynamics simulations of WT and E71A KcsA in heterogenous membranes of phosphatidylcholine and phosphatidylglycerol lipids of differing thickness supported by our experimental studies of electrophysiological activity and small angle X-ray scattering (SAXS).
343a
1693-Plat Insights into Ion Channel Selectivity with Ionic Coulomb Blockade William A.T. Gibby1, Dmitri G. Luchinsky2,3, Igor Kh Kaufman2, Peter V.E. McClintock2, Aneta Stefanovska2, Robert S. Eisenberg4. 1 University of Lancaster, Lancaster, United Kingdom, 2Department of Physics, University of Lancaster, Lancaster, United Kingdom, 3Mission Critical Technologies, El Segundo, CA, USA, 4Department of Molecular Biophysics, Rush University Medical Center, Chicago, IL, USA. The flow of ions through a biological ion channel can be considered as transitions between occupied energy levels in the channel and either of the connecting bulk reservoirs [2]. Discreteness of ions and an electrostatic exclusion principle ensure that the number of channel energy levels equals the number of occupying ions. Using these fundamental physical principles we have recently introduced [1] an ionic Coulomb blockade (ICB) theory developed by analogy with the similar phenomenon of electron tunnelling in quantum dots [2,3]. In this picture channel selectivity is governed by energy level changes [1]. We present details of the ICB theory for ion transitions through the channel. It incorporates physiological solutions and channel properties: physical dimension, voltage drop and fixed charge, and hence allows for comparison with physiological data. The set of kinetic equations obtained using ICB is analysed. The channel probability of occupancy as a function of transition rates (and hence fixed charge and number of ions) is obtained in the steady-state approximation. It is shown that this probability displays the staircase structure familiar from analysis of occupancy in quantum dots. It is also shown that current through the channel displays sharp peaks as a function of fixed charge, hence relating channel selectivity to the structure and position of energy levels. The contribution of hydration energy is also discussed. We anticipate that inclusion of this energy into ICB theory will provide an important insight into the selectivity and conductivity of ion channels. [1] Kaufman, I. K., McClintock, P. V. E., Eisenberg, R. S., New Journal of Physics. 17, 8, 15 (2015). [2] Beenakker, C.W.J., Phys. Rev. B., 44, 4, (1991). [3] Krems, M., Di Ventra, M., Phys. Condens. Matter, 25, 6, 065101 (2013). 1694-Plat Ionic Basis of Repolarization of Atrial and Ventricular Specific Cell Types Derived from Human Induced Pluripotent Stem Cells Aaron D. Kaplan1, Randall L. Rasmusson1, Glenna C.L. Bett2. 1 Physiology and Biophysics, Buffalo, NY, USA, 2OB/Gyn, Buffalo, NY, USA. Cardiomyocytes derived from human induced pluripotent stem cells (hIPSCCMs) are an innovative cellular system for understanding human cardiac pathology and physiology. However, hIPSC-CMs express low levels of inward rectifying potassium channel (IK1) relative to native cardiac cells. The lack of this current deforms the action potential (AP) and leads to a depolarized or spontaneous diastolic potential. We used electronic expression of IK1 via dynamic clamp to restore the resting membrane potential back to physiological levels. This resulted in an improved AP morphology, including a reduction in variability and a rate dependent spike and dome shape. There were several significant differences between atrial and ventricular cells including differences in cell capacitance, sodium current magnitude and kinetics and most prominently differences in the transient outward currents. The late components of outward current, cells with atrial APs had a significantly larger sustained outward IKUR or Kv1.5-like component at þ50 mV than ventricular shaped APs (in pA/pF: 3.71 5 0.55 (n=5) vs 1.00 5 0.10 (n=16),P<0.05) but similar peak outward currents: (6.89 5 0.50 (n=5) vs. 6.58 5 0 .67 (n=14),P=N.S). This plateau current is strongly inhibited by 50 micro molar 4-aminopyridine(4-AP). Similarly, atrial-like APs took on a ventricular like shape when treated with 4-AP while the ventricular myocytes APs were 4-AP insensitive. A cloned Kv1.5 current was expressed in oocytes and was used through the electronic expression system to add an IKur to the ventricular myocytes. Addition of this current changed the action potential morphology from ventricle to atrial like. This strongly suggests that IKur is the major determinant of atrial action potential morphology. 1695-Plat Shaker-IR K Channel Gating in Heavy Water: Role of Structural Water Molecules in Inactivation Tibor G. Szanto1, Szabolcs M. Gaal1, Zoltan Varga2, Gyorgy Panyi1. 1 Dept. of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary, 2MTA-DE-NAP B Ion Channel Structure-Function Research Group, University of Debrecen, Debrecen, Hungary. It has been reported earlier that the slow (C-type) inactivated conformation in eukaryotic KV channels is stabilized by a multipoint hydrogen-bond network
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behind the selectivity filter. Furthermore, the selectivity filter is sterically locked in the inactive conformation by buried water molecules and the binding of these ‘hidden’ molecules influence the inactivation process. We found that applying a heavy water (deuterium oxide, D2O)-based extracellular solution dramatically slowed the entry into the inactivated state which might indicate the role of the ‘‘structural water’’ molecules in the conformational stability of the selectivity filter. Alternatively, these observations can be explained by an increase in the viscosity or an altered residency time/exit rate of Kþ from the selectivity filter in a D2O-based extracellular solution. We mimicked the increased viscosity by adding glycerol to the extracellular solution and determined the inactivation kinetics in Shaker-IR channels having the following mutations and allowing slow inactivation at various rates: T449A and T449A/ I470A. The channels were transiently expressed in tsA_201 cells and ionic current experiments were recorded from either inside-out or outside-out patches. We found that application of 5% glycerol had negligible effect on the rate of inactivation kinetics. The exit rate of Kþ ions was studied by changing the Kþ gradient to allow the inactivation time constants to be determined for both outward and inward currents. Our results showed that exposure of the patches to extracellular D2O did not change the toutward/tinward ratio as compared to control (H2O on both sides). Therefore, our macroscopic current measurements support the hypothesis that structural water molecules may have specific effects on the inactivation kinetics by accessing to the region behind the selectivity filter, as proposed by molecular modelling data. 1696-Plat An Intrinsic Ligand Mediates N- and C-Terminal Interactions with the KCNH Gating Machinery Yaxian Zhao1, Joao H. Morais-Cabral2, Andreia Sousa Fernandes2, Gail A. Robertson1. 1 Department of Neuroscience, UW-Madison, Madison, WI, USA, 2Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal. EAG, ERG and ELK are members of the KCNH potassium channel family important in excitability. Two conserved domains in the cytosolic N and C termini uniquely characterize members of this family. The C-terminal domain has homology to the cyclic nucleotide-binding domain of CNG/HCN channels but is unaffected by cyclic nucleotides. At the tail end of this cyclic nucleotidebinding homology domain (CNBhD) is an intrinsic ligand comprising two highly conserved residues that loop back to occupy the cyclic nucleotidebinding pocket. The CNBhD interacts with the N-terminal domain, with one interface also occupied by the intrinsic ligand. We probed the intrinsic ligand and its effect on C- and N-terminal modulation of gating in hEAG1 channels. Using two-electrode voltage clamp of channels expressed in Xenopus oocytes, we found that substitution of the intrinsic ligand residues, Y699 and L701, with AA, SS and GG progressively shifted the midpoint of the conductance-voltage relationship to more depolarized potentials. The substitution with G, a residue unable to support a ligand-receptor interaction and thus considered fully unliganded, resulted in the greatest steady-state shift of channels into the closed state. Interestingly, deleting the CNBhD created the same phenotype as the GG mutant, suggesting the unliganded conformation confers a loss of function of the CNBhD’s effects on gating. Both AA and GG mutants abolished in vitro binding of the PAS domain to the CNBhD in anisotropy measurements. Functionally, when combining GG/CNBhD deletion with mutations at PAS-CNBhD interfaces, we observed a non-additive phenotype that is indistinguishable from the GG or CNBhD deletion mutant. These findings suggest that both the intrinsic ligand and CNBhD are required for the proper function of PAS domain, and the ligand and binding pocket can adopt multiple conformations, each conferring a different gating behavior. 1697-Plat Targeted Enhancement of hERG KD Channel Activity with scFv Antibody Fragments Greg Starek1, Carol A. Harley2, David K. Jones1, Gail A. Robertson1, Joa˜o H. Morais-Cabral2. 1 University of Wisconsin, Madison, WI, USA, 2IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal. Cardiac IKr is a repolarizing current comprising subunits encoded by the human ether-a-go-go related gene (hERG). A defining feature of hERG and related channel types is an N-terminal PAS domain that, through its effects on gating, reduces current amplitude. To probe the functional consequences of disrupting this natural channel modulation, we screened for single chain fragment variable (scFv) antibodies against the PAS domain using phage display. We assessed the binding properties to PAS and functional consequences of these antibodies on the hERG channel. Two isolates affecting hERG 1a current properties were shown by GST pull-down assays to interact with different regions of the PAS domain with submicromolar affinities. In
HEK-293 cells stably expressing hERG 1a, one antibody slowed inactivation onset and the other accelerated recovery from inactivation. Both significantly increased repolarizing current during a voltage protocol mimicking a ventricular action potential. Because native IKr channels are heteromers of the original hERG 1a isolate and hERG 1b, an isoform from which the PAS domain is transcriptionally omitted, we tested the effect of the scFv antibodies on cardiomyocytes derived from human induced pluripotent stem cells. Similar to the HEK-293 findings, the antibodies diminished channel rectification and significantly increased the repolarizing charge during a ventricular action potential voltage clamp. Here, we demonstrate the versatility of scFv antibodies as a site-specific probe of ion channel function. Given their ability to increase IKr, we propose the PAS-channel interface as a therapeutic target and the use of scFv antibodies as a novel treatment for diseases of excitability where action potential duration is prolonged. 1698-Plat Functional and Crystallographic Studies of Hetero-Multimeric KD Channels Spandana Vemulapally, D. Marien Cortes, Luis G. Cuello. Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Science Center, Lubbock, TX, USA. Voltage gated potassium channels (Kv channels) consist of four identical subunits that form a central permeation pathway in which changes in diameter of an inner constriction known as the activation gate (AG) catalyzes their transition from the closed to the open state. Kv channels play a major role in shaping and regulating the action potential in all excitable cells. Expressed as homotetramers as well as heterotetramers, Kv channels have a crucial role in excitability disorders such as arrhythmias, long QT syndrome, hyperactivity disorders, epilepsia and many other behavioral disorders. This renders heteromeric Kv channels as potential pharmaceutical targets but challenges arise because of their hetero-multimeric nature and diverse sequence identity, which make the smart design of safer and more specific therapeutic drugs difficult. Thus, it is of significant importance to study in detail the structure-function relationship in heteromeric Kv channels. Since KcsA is the bona fide structural and functional surrogate of the pore domain of Kv channels, we have constructed tandem-tetramer constructs of KcsA in which all four protomers are expressed as a single polypeptide chain by engineering linkers between the protomers. We were able to express, purify and solved the crystal structure of functional KcsA tandem-tetramer. This opens up a new and unprecedented avenue to introduce dose dependent mutations in the channel tetramer to evaluate the cooperative behavior of the channel function i.e., ion permeation, gating and ion selectivity, from a functional and structural point of view by using electrophysiology and X-ray crystallography approaches. Founding: AHA-11SDG5440003, NIH 1RO1GMo97159-01A1 and Welch Foundation BI-1757 1699-Plat Optical Recording of Voltage Activation of Endogenous Potassium Channels Mark W. Lillya1, Laxmi K. Parajuli2, Sebastian Fletcher-Taylor1, Joyce Huang1, Bruce E. Cohen3, Karen Zito2, Jon T. Sack1. 1 Physiology and Membrane Biology, University of California, Davis, Davis, CA, USA, 2Center for Neuroscience, University of California, Davis, Davis, CA, USA, 3Biological Nanostructures Facility, Laurence Berkeley National Laboratory, Berkeley, CA, USA. Diverse cellular electrical signals are conducted by a vast array of ion channel types acting in concert. The spatial and temporal contributions of specific voltage gated ion channels to neuronal electrical signaling are challenging to establish. We have developed fluorescent probes that indicate when and where Kv2 voltage gated potassium channels are activated by voltage. These ion channel activity probes are engineered from tarantula toxins that bind Kv2 channel voltage sensors at rest, and dissociate when the channels are voltage activated. Previously, we found ion channel activity probes to detect overexpressed Kv2 ion channels. Here, we explore their ability to identify activation of endogenous neuronal ion channels. In rat hippocampal slices and dissociated neurons, we find that exogenously applied Kv2 ion channel probes label punctate structures on neuronal cell bodies similar to endogenous Kv2 ion channels. In neurons transfected with GFP-Kv2.1, ion channel probe puncta colocalize with GFP puncta. Depolarizing stimuli that induce Kv2 channel activation, trigger dissipation of fluorescent puncta from neurons. Together, our results suggest that ion channel probes can identify the locations of specific ion channel types in living neurons, and report when and where the channels are activated by voltage. Further development of probes that target to select conformations of ion channels could enable imaging of the contributions of specific ion channel types to electrical activity in neuronal networks.