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Effect of 2-Aminoethylmethanethiosulphonate (MTSEA) on the HKV1.3_L346C and the HKV1.3_L346C_L418C Mutant Channels
278a
Monday, February 29, 2016
Voltage-sensing modules (VSMs) of similar molecular structure underlie voltage-sensitivity in ion channels, the voltage-sensor of EC coupling (the DHPR), and voltage-sensitive phosphatases (VSPs). Upon sustained depolarization (or ‘‘antipolarization’’ of VSPs) all VSMs enter a functionally disabled inactivated or relaxed state. This is accompanied in every case by a change in voltage dependence of the sensor charge movement, including a large negative shift of its central voltage VT. In frog muscle, voltage-driven transitions of the EC coupling VSM elicit charge movements that result in two charge distributions with different VT: Q1 in the normally polarized fiber; Q2 when the membrane is held depolarized. Here we show in mammalian muscle —single fibers of mouse FDB— a similar interconversion of voltage-sensor charge. In 2 mouse strains VT at rest was ~-20 mV, but shifted to ~-80 mV upon sustained depolarization. The change was a conversion between two distributions (Q1 and Q2) with distinct but fixed VT‘s. The observations require a minimum model with 4 states: C (cis), T (trans), CR (cis relaxed), TR (trans relaxed). The transitions C->T and CR->TR are fast and mobilize respectively Q1 and Q2. C->T opens the RyR, but CR->TR does not. The transitions to and from relaxed states are slow and voltage-independent. Low extracellular [Ca2D] promoted the C->CR and T->TR transitions, while high [Ca2D] opposed it. Observations of similar antagonism between inactivation of Ca2D and KD channels and the pore occupancy by ions led to the notion that inactivation involves pore collapse. DHPR pore opening, however, is not required for proper operation of the EC coupling VSM. The effects of extracellular Ca2D on voltage-dependent inactivation must be reinterpreted in view of these findings. (Support: ANII, CSIC and NIGMS). 1369-Pos Board B346 Effect of 2-Aminoethylmethanethiosulphonate (MTSEA) on the HKV1.3_L346C and the HKV1.3_L346C_L418C Mutant Channels Ann-Kathrin Diesch, Stephan Grissmer. Institute of Applied Physiology, Ulm, Germany. The movement of some residues in the inner and outer pore region of the hKv1.3 channel causes its characteristic C-type inactivation. To get more details about the spatial arrangement of the inactivated state, we introduced cysteine mutations and assessed the influence on the current by applying extracellular MTSEA, a cysteine modifying reagent. Former studies showed that structural changes of the inner and outer pore during gating had an effect on the accessibility of the cysteine side chains. Amino acids L418C and T419C (Shaker positions 468, 469) in the S6 segment could only be modified by MTSEA in the open but not in the inactivated or closed state whereas the cysteines at positions V417C and I420C (Shaker positions 467, 470) could be modified in the open and inactivated state and not in the closed state. In an attempt to find those structures of the channel that prevented MTS modification in the inactivated L418C mutant channel we examined the L346C (Shaker positions 396) in the S5 segment which is in close proximity to L418. Currents through hKv1.3_L346C channels had similar characteristics as currents through wild-type hKv1.3 channels and could –like wild type channels- not be modified by externally applied MTSEA. The double mutant hKv1.3_L346C_L418C channel could be modified by MTSEA in the open but not in the inactivated and closed state indicating that the L346C mutation does not alter the modification behavior of hKv1.3_L418C. We conclude that the relative movement of the S6 segments that occurring during C-type inactivation that also includes a movement of the side chains of the amino acid at position 418 in hKv1.3 channels was not disturbed by the L346C mutation. This work was supported by a grant from the DFG (Gr848/17-1). 1370-Pos Board B347 Elucidation of Molecular Mechanism Underlying KcsA’s Hysteretic Gating Behavior Cholpon Tilegenova, D. Marien Cortes, Luis G. Cuello. Cell Physiology and Molecular Biophysics, TTUHSC, Lubbock, TX, USA. Hysteresis has been observed in cyclic nucleotide-gated4, transient receptor potential vanilloid3, N-methyl-D-aspartate5, human Ether-a`-go-go-Related Gene6, human HCN48, mouse HCN11 channels. Voltage shift for QV (gating charge vs voltage) curve is well documented for voltage-gated cation channels, which was considered an intrinsic property of the voltage-sensing domain (VSD). However, it was showed recently that uncoupling the VSD from the pore domain of the Shaker Kþ channel, eliminated the VSD’s hysteretic gating behavior. It was suggested that the pore domain imposes a mechanical load on the VSD due to stabilization of an open state, which causes the hysteresis2. Since open-state stabilization occur at the channels’ pore domain, it is important to study the pore domain gating mechanism in isolation. We intend to use the archetypal pore domain of a Kþ channel, KcsA, as a functional and structural surrogate, to elucidate the molecular basis of hysteretic gating behavior in ion channels. Recently, we have unveiled a novel hysteretic gating behavior in
KcsA by electrophysiology and by continuous wave electron paramagnetic resonance spectroscopy, which faithfully measure the pH dependent conformational changes associated to activation7 and deactivation gating. We hypothesize that structural changes of the KcsA’s selectivity filter associated to C-type inactivation underlie the molecular mechanism of hysteretic gating in KcsA and by extension in other ion channels. The long-term goal of this project is to determine the molecular basis for hysteretic gating in ion channels, which can be useful for the development of drugs that can correct several channelopathies. 1371-Pos Board B348 Ginsenoside Rg3 Activates Human EAG Family of KD Channels via Allosteric Modification of Gating Wei Wu, Alison Gardner, Michael Sanguinetti. University of Utah, Salt Lake City, UT, USA. The human ether-a`-go-go (EAG) family of voltage-gated Kþ channels, including hERG, hEAG, and hELK exhibit variable biophysical and physiological properties. Ginsenoside Rg3 (Rg3), a steroid glycoside is a potent hERG channel activator. Here we characterize the mechanisms of action of Rg3 on three EAG-family representative channels, including hERG1, hEAG1 and hELK1 heterologously expressed in Xenopus laevis oocytes. Rg3 enhanced hERG1 channel current via a negative shift in the half-point for the voltage dependence of activation (V0.5act) that saturated at 14 mV, and a profound slowing in the rate of deactivation (EC50 = 242 nM). Rg3 had no effect on the rate of activation or the voltage dependence of hERG1 inactivation gating. For hEAG1 channels, Rg3 induced a 28 mV maxiumal shift in V0.5act (EC50 = 1.18 mM) and accelerated the rate of activation (~20-fold faster). The maximum shift in V0.5act for hELK1 channels was much greater (>-100 mV; EC50 = 176 nM). The onset of Rg3 effects upon extracellular application was very fast and rapidly reversible upon washout for all 3 channel types, indicating this large molecule (MW = 785) binds to an extracellular region. Rg3 at 3 mM had no effect on Kv1.5 channels and only affects other Kv channels at much higher concentrations. Understanding the mechanisms of action of Rg3 may facilitate the development of more potent and selective gensenosides for therapeutic use. 1372-Pos Board B349 hERG S4-S5 acts as a Voltage-Dependent Ligand Binding the Activation Gate and Locking it in a Closed State Olfat Malak, Gildas Loussouarn, Zeineb Es-Salah-Lamoureux. l’institut du thorax, Nantes, France. Kv (voltage-gated potassium) channels are formed by a tetrameric pore (S5–S6) surrounded by four voltage sensor domains (S1–S4). Molecular mechanisms underlying gating remain poorly understood.Previously, we showed that peptides mimicking S4-S5L inhibit a cardiac Kv channel, KCNQ1 (Kv7.1). On the other hand, peptides mimicking S6T upregulate the channel activity by competing with the endogenous S6T for the endogenous S4-S5L (Choveau et al, 2010). Those observations suggested that S4-S5L acts as a voltage controled ligand that binds S6T and locks the channel in a closed state. However, given the low affinity between the peptide and the KCNQ1 channel, effects were moderate. In the present study, we asked whether covalently locking s4s5l onto s6t would lead to a permanent closure of the channel. To this end, we selected the human ether-a-go-go-related gene (hERG or Kv11.1) channel as a model since in this channel, a couple of cysteine in S4-S5L and S6T was introduced and led to channel closure (Ferrer et al, 2006). We used the whole-cell configuration of the patch-clamp technique in transfected COS-7 cells. We observed a complete inhibition of hERG in oxidative condition when coexpressing the S4-S5L peptide carrying an introduced cysteine (S4S5L-Cys-peptide) and the channel with a cysteine introduced in S6T (S6T-Cys-channel). Reciprocally, coexpressing S6TCyspeptide and the S4-S5L-Cys-channel led to a virtually voltage independent channel in oxidative condition. These results: 1) validate a model of gating in which S4-S5 acts as a voltage-dependent ligand binding the activation gate and locking it in a closed state and 2) suggest a generalization of this model. Choveau et al, J Biol Chem. 2011 286:707-16 Ferrer et al, J Biol Chem. 2006 281:12858-64 1373-Pos Board B350 7-Dehydrocholesterol Modifies the Operation of Kv1.3 Channels in T Cells Isolated from Smith-Lemli-Opitz Syndrome Patients Andras Balajthy1, Zoltan Petho1, Sandor Somodi2, Zoltan Varga1, Maria Peter3, Laszlo Vı´gh3, Gabriella P. Szabo´4, Gyorgy Paragh2, Gyorgy Panyi1, Peter Hajdu1. 1 Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary, 2 Department of Internal Medicine, University of Debrecen, Debrecen, Hungary, 3Laboratory of Molecular Stress Biology, Biological Research Centre, Szeged, Hungary, 4Department of Pediatrics, University of Debrecen, Debrecen, Hungary.
Monday, February 29, 2016
Voltage-sensing modules (VSMs) of similar molecular structure underlie voltage-sensitivity in ion channels, the voltage-sensor of EC coupling (the DHPR), and voltage-sensitive phosphatases (VSPs). Upon sustained depolarization (or ‘‘antipolarization’’ of VSPs) all VSMs enter a functionally disabled inactivated or relaxed state. This is accompanied in every case by a change in voltage dependence of the sensor charge movement, including a large negative shift of its central voltage VT. In frog muscle, voltage-driven transitions of the EC coupling VSM elicit charge movements that result in two charge distributions with different VT: Q1 in the normally polarized fiber; Q2 when the membrane is held depolarized. Here we show in mammalian muscle —single fibers of mouse FDB— a similar interconversion of voltage-sensor charge. In 2 mouse strains VT at rest was ~-20 mV, but shifted to ~-80 mV upon sustained depolarization. The change was a conversion between two distributions (Q1 and Q2) with distinct but fixed VT‘s. The observations require a minimum model with 4 states: C (cis), T (trans), CR (cis relaxed), TR (trans relaxed). The transitions C->T and CR->TR are fast and mobilize respectively Q1 and Q2. C->T opens the RyR, but CR->TR does not. The transitions to and from relaxed states are slow and voltage-independent. Low extracellular [Ca2D] promoted the C->CR and T->TR transitions, while high [Ca2D] opposed it. Observations of similar antagonism between inactivation of Ca2D and KD channels and the pore occupancy by ions led to the notion that inactivation involves pore collapse. DHPR pore opening, however, is not required for proper operation of the EC coupling VSM. The effects of extracellular Ca2D on voltage-dependent inactivation must be reinterpreted in view of these findings. (Support: ANII, CSIC and NIGMS). 1369-Pos Board B346 Effect of 2-Aminoethylmethanethiosulphonate (MTSEA) on the HKV1.3_L346C and the HKV1.3_L346C_L418C Mutant Channels Ann-Kathrin Diesch, Stephan Grissmer. Institute of Applied Physiology, Ulm, Germany. The movement of some residues in the inner and outer pore region of the hKv1.3 channel causes its characteristic C-type inactivation. To get more details about the spatial arrangement of the inactivated state, we introduced cysteine mutations and assessed the influence on the current by applying extracellular MTSEA, a cysteine modifying reagent. Former studies showed that structural changes of the inner and outer pore during gating had an effect on the accessibility of the cysteine side chains. Amino acids L418C and T419C (Shaker positions 468, 469) in the S6 segment could only be modified by MTSEA in the open but not in the inactivated or closed state whereas the cysteines at positions V417C and I420C (Shaker positions 467, 470) could be modified in the open and inactivated state and not in the closed state. In an attempt to find those structures of the channel that prevented MTS modification in the inactivated L418C mutant channel we examined the L346C (Shaker positions 396) in the S5 segment which is in close proximity to L418. Currents through hKv1.3_L346C channels had similar characteristics as currents through wild-type hKv1.3 channels and could –like wild type channels- not be modified by externally applied MTSEA. The double mutant hKv1.3_L346C_L418C channel could be modified by MTSEA in the open but not in the inactivated and closed state indicating that the L346C mutation does not alter the modification behavior of hKv1.3_L418C. We conclude that the relative movement of the S6 segments that occurring during C-type inactivation that also includes a movement of the side chains of the amino acid at position 418 in hKv1.3 channels was not disturbed by the L346C mutation. This work was supported by a grant from the DFG (Gr848/17-1). 1370-Pos Board B347 Elucidation of Molecular Mechanism Underlying KcsA’s Hysteretic Gating Behavior Cholpon Tilegenova, D. Marien Cortes, Luis G. Cuello. Cell Physiology and Molecular Biophysics, TTUHSC, Lubbock, TX, USA. Hysteresis has been observed in cyclic nucleotide-gated4, transient receptor potential vanilloid3, N-methyl-D-aspartate5, human Ether-a`-go-go-Related Gene6, human HCN48, mouse HCN11 channels. Voltage shift for QV (gating charge vs voltage) curve is well documented for voltage-gated cation channels, which was considered an intrinsic property of the voltage-sensing domain (VSD). However, it was showed recently that uncoupling the VSD from the pore domain of the Shaker Kþ channel, eliminated the VSD’s hysteretic gating behavior. It was suggested that the pore domain imposes a mechanical load on the VSD due to stabilization of an open state, which causes the hysteresis2. Since open-state stabilization occur at the channels’ pore domain, it is important to study the pore domain gating mechanism in isolation. We intend to use the archetypal pore domain of a Kþ channel, KcsA, as a functional and structural surrogate, to elucidate the molecular basis of hysteretic gating behavior in ion channels. Recently, we have unveiled a novel hysteretic gating behavior in
KcsA by electrophysiology and by continuous wave electron paramagnetic resonance spectroscopy, which faithfully measure the pH dependent conformational changes associated to activation7 and deactivation gating. We hypothesize that structural changes of the KcsA’s selectivity filter associated to C-type inactivation underlie the molecular mechanism of hysteretic gating in KcsA and by extension in other ion channels. The long-term goal of this project is to determine the molecular basis for hysteretic gating in ion channels, which can be useful for the development of drugs that can correct several channelopathies. 1371-Pos Board B348 Ginsenoside Rg3 Activates Human EAG Family of KD Channels via Allosteric Modification of Gating Wei Wu, Alison Gardner, Michael Sanguinetti. University of Utah, Salt Lake City, UT, USA. The human ether-a`-go-go (EAG) family of voltage-gated Kþ channels, including hERG, hEAG, and hELK exhibit variable biophysical and physiological properties. Ginsenoside Rg3 (Rg3), a steroid glycoside is a potent hERG channel activator. Here we characterize the mechanisms of action of Rg3 on three EAG-family representative channels, including hERG1, hEAG1 and hELK1 heterologously expressed in Xenopus laevis oocytes. Rg3 enhanced hERG1 channel current via a negative shift in the half-point for the voltage dependence of activation (V0.5act) that saturated at 14 mV, and a profound slowing in the rate of deactivation (EC50 = 242 nM). Rg3 had no effect on the rate of activation or the voltage dependence of hERG1 inactivation gating. For hEAG1 channels, Rg3 induced a 28 mV maxiumal shift in V0.5act (EC50 = 1.18 mM) and accelerated the rate of activation (~20-fold faster). The maximum shift in V0.5act for hELK1 channels was much greater (>-100 mV; EC50 = 176 nM). The onset of Rg3 effects upon extracellular application was very fast and rapidly reversible upon washout for all 3 channel types, indicating this large molecule (MW = 785) binds to an extracellular region. Rg3 at 3 mM had no effect on Kv1.5 channels and only affects other Kv channels at much higher concentrations. Understanding the mechanisms of action of Rg3 may facilitate the development of more potent and selective gensenosides for therapeutic use. 1372-Pos Board B349 hERG S4-S5 acts as a Voltage-Dependent Ligand Binding the Activation Gate and Locking it in a Closed State Olfat Malak, Gildas Loussouarn, Zeineb Es-Salah-Lamoureux. l’institut du thorax, Nantes, France. Kv (voltage-gated potassium) channels are formed by a tetrameric pore (S5–S6) surrounded by four voltage sensor domains (S1–S4). Molecular mechanisms underlying gating remain poorly understood.Previously, we showed that peptides mimicking S4-S5L inhibit a cardiac Kv channel, KCNQ1 (Kv7.1). On the other hand, peptides mimicking S6T upregulate the channel activity by competing with the endogenous S6T for the endogenous S4-S5L (Choveau et al, 2010). Those observations suggested that S4-S5L acts as a voltage controled ligand that binds S6T and locks the channel in a closed state. However, given the low affinity between the peptide and the KCNQ1 channel, effects were moderate. In the present study, we asked whether covalently locking s4s5l onto s6t would lead to a permanent closure of the channel. To this end, we selected the human ether-a-go-go-related gene (hERG or Kv11.1) channel as a model since in this channel, a couple of cysteine in S4-S5L and S6T was introduced and led to channel closure (Ferrer et al, 2006). We used the whole-cell configuration of the patch-clamp technique in transfected COS-7 cells. We observed a complete inhibition of hERG in oxidative condition when coexpressing the S4-S5L peptide carrying an introduced cysteine (S4S5L-Cys-peptide) and the channel with a cysteine introduced in S6T (S6T-Cys-channel). Reciprocally, coexpressing S6TCyspeptide and the S4-S5L-Cys-channel led to a virtually voltage independent channel in oxidative condition. These results: 1) validate a model of gating in which S4-S5 acts as a voltage-dependent ligand binding the activation gate and locking it in a closed state and 2) suggest a generalization of this model. Choveau et al, J Biol Chem. 2011 286:707-16 Ferrer et al, J Biol Chem. 2006 281:12858-64 1373-Pos Board B350 7-Dehydrocholesterol Modifies the Operation of Kv1.3 Channels in T Cells Isolated from Smith-Lemli-Opitz Syndrome Patients Andras Balajthy1, Zoltan Petho1, Sandor Somodi2, Zoltan Varga1, Maria Peter3, Laszlo Vı´gh3, Gabriella P. Szabo´4, Gyorgy Paragh2, Gyorgy Panyi1, Peter Hajdu1. 1 Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary, 2 Department of Internal Medicine, University of Debrecen, Debrecen, Hungary, 3Laboratory of Molecular Stress Biology, Biological Research Centre, Szeged, Hungary, 4Department of Pediatrics, University of Debrecen, Debrecen, Hungary.