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KvAP Gating States Probed by Electron Spin-Echo Envelope Modulation (ESEEM) Spectroscopy
Wednesday, March 2, 2016 2965-Pos Board B342 Sildenafil is Effective to Enhance the Proliferation of Skeletal Myoblasts Mei Huang1, Keon Jin Lee1, Mi Kyoung Ahn1, Chung-Hyun Cho2, Eun Hui Lee1. 1 Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea, Seoul, Korea, Republic of, 2Dept. of Pharmacology, College of Medicine, Seoul National University, Seoul, Korea, Republic of. Sildenafil is a specific inhibitor of phosphodiesterase type 5 and is clinically used to treat erectile dysfunction and pulmonary artery hypertension because of its vasodilatation effect due to the relaxation of smooth muscle cells. In cardiac muscle, the cardioprotective effects of sildenafil have been reported. The effectiveness of sildenafil on skeletal muscle has been controversial: sildenafil reduces skeletal muscle fatigue, however, the atrophy of skeletal muscle by sildenafil is also reported. In addition, the effect of sildenafil on skeletal muscle in cellular levels has not been examined. In the present study, sildenafil effect on skeletal muscle cells was examined using mouse primary skeletal myoblasts. Sildenafil was effective to enhance the proliferation of the skeletal myoblasts.
Voltage-gated K Channels, Mechanisms of Voltage Sensing and Gating III 2966-Pos Board B343 Tryptophan 207 is Crucial to the Unique Properties of the Human Voltage Gated Proton Channel, hHv1 Vladimir V. Cherny1, Deri Morgan1, Boris Musset2, Gustavo Chaves2, Susan M.E. Smith3, Thomas E. DeCoursey1. 1 Molecular Biophysics & Physiology, Rush University, Chicago, IL, USA, 2 Institute of Complex Systems (ICS-4 Zellula¨re Biophysik), Forschungszentrum Ju¨lich, Ju¨lich, Germany, 3Biology and Physics, Kennesaw State University, Kennesaw, GA, USA. Part of the ‘‘signature sequence’’ that defines voltage-gated proton channels (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, that is perfectly conserved in all high-confidence HV1 genes. Replacing Trp207 in human HV1 with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than WT channels, thus in WT channels Trp favors a closed state. The Ea for channel opening decreased to 22 kcal/mol from 30-38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hHV1. Cation-p interaction between Trp207 and Arg211 latches the channel closed. Trp207 mutants lost proton selectivity at pHo > 8. Finally, DpH dependent gating, a universal feature of HV1 that is essential to its biological functions, was compromised. In the WT hHV1 channel DpH dependent gating saturates above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of DpH, indicating distinct internal and external pH sensors. In Trp207 mutants DpH dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in HV1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of DpH dependent gating occurred at the same pHo and pHi in HV1 of all three species, suggesting that the same or similar group(s) are involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature dependent gating kinetics, proton selectivity, and DpH dependent gating. 2967-Pos Board B344 Hv1 Proton Channel Resting-State Voltage Sensor Model Structures are Refined by Experimental Mapping of Zinc-Coordinating Residues Victor De-la-Rosa, Ashley L. Bennett, Ian Scott Ramsey. Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, USA. Voltage sensor (VS) activation likely involves an outward movement of the S4 helix from its resting state conformation, but high resolution resting-state VS structures are rare and experimental data is needed to spatially constrain model structures. Zn2þ potently (EC50 z 1 uM, pHO 6.5) attenuates Hþ currents in the human voltage-gated proton channel (hHv1) by shifting the apparent POPEN-V relation toward more positive potentials (Cherny, et al. 1999). Zn2þ is likely to be coordinated by two extracellularly-accessible His residues, H140 (S2 helix) and H193 (S3-S4 linker) in the resting conformation of the Hv1 VS domain (Ramsey et al., 2006). The intermediate potency (EC50 20-70 uM) of Zn2þ in single H140A or H193A mutant channels suggests that additional residues may also participate resting-state Zn2þ coordination. Candidate residues include the acidic side chains of E119 (S1) and D123 (S1-S2 linker), which
601a
may be near H140 and H193 in the mouse Hv1-based voltage sensor chimeric protein mHv1cc (Takeshita et al., 2014). To identify Zn2þ-coordinating residues in hHv1, we mutated candidate acidic residues to His in the background of either H140A or H193A and measured the potency of Zn2þ to attenuate voltage-gated Hþ currents. We find that E119H, but not D123H or D130H, is sufficient to reconstitute potent (low micromolar EC50) block of current by Zn2þ when combined with either H193A or H140A, indicating that E119 is likely to coordinate Zn2þ in conjunction with H140 and H193 in WT hHv1. Our experimental data limit the distances of E119, H140 and H193 side chains from the coordinated Zn2þ ion and therefore strongly constrain the positions of these groups, allowing us to generate refined resting-state hHv1 VS model structures. 2968-Pos Board B345 KvAP Gating States Probed by Electron Spin-Echo Envelope Modulation (ESEEM) Spectroscopy Dylan O. Burdette, Adrian Gross. Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA. During a gating event, the voltage gated potassium channel KvAP undergoes a conformational change in the voltage sensor (helices 1-4), causing the opening of the pore domain (helices 5-6). Throughout this conformational change, the extracellular portion of the pore remains relatively immobile. However, the environment surrounding this part of the pore domain may be altered through changes in lipid packing or tertiary contacts within the channel. In this study, model membranes were used to stabilize KvAP in different gating states and the environment surrounding the pore were probed with ESEEM both in the presence and absence of the voltage sensing domain. 2969-Pos Board B346 Regulation of hERG1b by hERG1a N-Terminal Regions Beth A. McNally, Matthew C. Trudeau. University of Maryland Baltimore, Baltimore, MD, USA. The human Ether-a´-go-go-Related Gene (hERG) encodes a voltage-activated potassium channel. Two hERG isoforms, hERG1a and hERG1b, play a critical role in the repolarization of the ventricular action potential in the heart and form the rapid component of the delayed rectifier potassium current, known as IKr. The presence of both hERG1a and hERG1b at the plasma membrane is critical for normal IKr, which is dependent on hERG1a and hERG1b protein assembly and interaction. However, it is unknown how hERG1a and hERG1b assemble and interact. Here, we used a variety of experimental techniques including electrophysiology, biotinylation, protein interaction assays, FRET microscopy, and protein engineering to study the hERG1a and hERG1b interaction. We have identified hERG1a N-terminal regions that selectively increase hERG1b current by increasing hERG1b protein at the plasma membrane. This effect is specific to hERG1b as hERG1a N-terminal regions do not increase hERG1a current. In addition, our FRET and protein interaction studies support a direct interaction between the hERG1a N-terminal region and the unique hERG1b N-terminal region. Together these results help to elucidate the structural requirements underscoring the hERG1a and hERG1b interaction. 2970-Pos Board B347 Multiple Metal Bridges at the Intracellular Gate of a Voltage Activated Potassium Channel Prevent Closing Angel A. de la Cruz Landrau1,2, Miguel Holmgren3. 1 National Intitute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA, 2Cell and Molecular Biology, Universidad Central del Caribe, Bayamo´n, PR, USA, 3NINDS, NIH, Bethesda, MD, USA. Voltage-gated potassium (Kv) channels control Kþ permeation in excitable cells. Kv channels are tetramers of a subunit, each composed of six transmembrane segments (S1-S6). The S5, P-loop and the S6 segments form the channel’s permeation pathway. At the inner end of the S6, an intracellular gate opens or closes in response to voltage. A valine to cysteine mutation at position 476, near the gate, traps the mutant channel in the open state when Cd2þ is added intracellularly. It has been previously shown that four metal bridges are formed between the cysteine at position 476 of one subunit and a native histidine at position 486 in an adjacent subunit. To understand the contribution of individual bridges, we constructed a concatemer Kv channel with all subunits linked at the DNA level. After introducing the mutation V476C, ionic currents were measured using excised inside-out patches to access the intracellular part of the channel. In the absence of Cd2þ, V476C concatemer mutants open and close normally. In the presence of Cd2þ, successive addition of V476C mutations to the subunits of the concatemer increasingly slowed down the closing of the channel at 120 mV. With all four V476C mutations, channels are locked open with Cd2þ.
Voltage-gated K Channels, Mechanisms of Voltage Sensing and Gating III 2966-Pos Board B343 Tryptophan 207 is Crucial to the Unique Properties of the Human Voltage Gated Proton Channel, hHv1 Vladimir V. Cherny1, Deri Morgan1, Boris Musset2, Gustavo Chaves2, Susan M.E. Smith3, Thomas E. DeCoursey1. 1 Molecular Biophysics & Physiology, Rush University, Chicago, IL, USA, 2 Institute of Complex Systems (ICS-4 Zellula¨re Biophysik), Forschungszentrum Ju¨lich, Ju¨lich, Germany, 3Biology and Physics, Kennesaw State University, Kennesaw, GA, USA. Part of the ‘‘signature sequence’’ that defines voltage-gated proton channels (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, that is perfectly conserved in all high-confidence HV1 genes. Replacing Trp207 in human HV1 with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than WT channels, thus in WT channels Trp favors a closed state. The Ea for channel opening decreased to 22 kcal/mol from 30-38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hHV1. Cation-p interaction between Trp207 and Arg211 latches the channel closed. Trp207 mutants lost proton selectivity at pHo > 8. Finally, DpH dependent gating, a universal feature of HV1 that is essential to its biological functions, was compromised. In the WT hHV1 channel DpH dependent gating saturates above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of DpH, indicating distinct internal and external pH sensors. In Trp207 mutants DpH dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in HV1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of DpH dependent gating occurred at the same pHo and pHi in HV1 of all three species, suggesting that the same or similar group(s) are involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature dependent gating kinetics, proton selectivity, and DpH dependent gating. 2967-Pos Board B344 Hv1 Proton Channel Resting-State Voltage Sensor Model Structures are Refined by Experimental Mapping of Zinc-Coordinating Residues Victor De-la-Rosa, Ashley L. Bennett, Ian Scott Ramsey. Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, USA. Voltage sensor (VS) activation likely involves an outward movement of the S4 helix from its resting state conformation, but high resolution resting-state VS structures are rare and experimental data is needed to spatially constrain model structures. Zn2þ potently (EC50 z 1 uM, pHO 6.5) attenuates Hþ currents in the human voltage-gated proton channel (hHv1) by shifting the apparent POPEN-V relation toward more positive potentials (Cherny, et al. 1999). Zn2þ is likely to be coordinated by two extracellularly-accessible His residues, H140 (S2 helix) and H193 (S3-S4 linker) in the resting conformation of the Hv1 VS domain (Ramsey et al., 2006). The intermediate potency (EC50 20-70 uM) of Zn2þ in single H140A or H193A mutant channels suggests that additional residues may also participate resting-state Zn2þ coordination. Candidate residues include the acidic side chains of E119 (S1) and D123 (S1-S2 linker), which
601a
may be near H140 and H193 in the mouse Hv1-based voltage sensor chimeric protein mHv1cc (Takeshita et al., 2014). To identify Zn2þ-coordinating residues in hHv1, we mutated candidate acidic residues to His in the background of either H140A or H193A and measured the potency of Zn2þ to attenuate voltage-gated Hþ currents. We find that E119H, but not D123H or D130H, is sufficient to reconstitute potent (low micromolar EC50) block of current by Zn2þ when combined with either H193A or H140A, indicating that E119 is likely to coordinate Zn2þ in conjunction with H140 and H193 in WT hHv1. Our experimental data limit the distances of E119, H140 and H193 side chains from the coordinated Zn2þ ion and therefore strongly constrain the positions of these groups, allowing us to generate refined resting-state hHv1 VS model structures. 2968-Pos Board B345 KvAP Gating States Probed by Electron Spin-Echo Envelope Modulation (ESEEM) Spectroscopy Dylan O. Burdette, Adrian Gross. Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA. During a gating event, the voltage gated potassium channel KvAP undergoes a conformational change in the voltage sensor (helices 1-4), causing the opening of the pore domain (helices 5-6). Throughout this conformational change, the extracellular portion of the pore remains relatively immobile. However, the environment surrounding this part of the pore domain may be altered through changes in lipid packing or tertiary contacts within the channel. In this study, model membranes were used to stabilize KvAP in different gating states and the environment surrounding the pore were probed with ESEEM both in the presence and absence of the voltage sensing domain. 2969-Pos Board B346 Regulation of hERG1b by hERG1a N-Terminal Regions Beth A. McNally, Matthew C. Trudeau. University of Maryland Baltimore, Baltimore, MD, USA. The human Ether-a´-go-go-Related Gene (hERG) encodes a voltage-activated potassium channel. Two hERG isoforms, hERG1a and hERG1b, play a critical role in the repolarization of the ventricular action potential in the heart and form the rapid component of the delayed rectifier potassium current, known as IKr. The presence of both hERG1a and hERG1b at the plasma membrane is critical for normal IKr, which is dependent on hERG1a and hERG1b protein assembly and interaction. However, it is unknown how hERG1a and hERG1b assemble and interact. Here, we used a variety of experimental techniques including electrophysiology, biotinylation, protein interaction assays, FRET microscopy, and protein engineering to study the hERG1a and hERG1b interaction. We have identified hERG1a N-terminal regions that selectively increase hERG1b current by increasing hERG1b protein at the plasma membrane. This effect is specific to hERG1b as hERG1a N-terminal regions do not increase hERG1a current. In addition, our FRET and protein interaction studies support a direct interaction between the hERG1a N-terminal region and the unique hERG1b N-terminal region. Together these results help to elucidate the structural requirements underscoring the hERG1a and hERG1b interaction. 2970-Pos Board B347 Multiple Metal Bridges at the Intracellular Gate of a Voltage Activated Potassium Channel Prevent Closing Angel A. de la Cruz Landrau1,2, Miguel Holmgren3. 1 National Intitute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA, 2Cell and Molecular Biology, Universidad Central del Caribe, Bayamo´n, PR, USA, 3NINDS, NIH, Bethesda, MD, USA. Voltage-gated potassium (Kv) channels control Kþ permeation in excitable cells. Kv channels are tetramers of a subunit, each composed of six transmembrane segments (S1-S6). The S5, P-loop and the S6 segments form the channel’s permeation pathway. At the inner end of the S6, an intracellular gate opens or closes in response to voltage. A valine to cysteine mutation at position 476, near the gate, traps the mutant channel in the open state when Cd2þ is added intracellularly. It has been previously shown that four metal bridges are formed between the cysteine at position 476 of one subunit and a native histidine at position 486 in an adjacent subunit. To understand the contribution of individual bridges, we constructed a concatemer Kv channel with all subunits linked at the DNA level. After introducing the mutation V476C, ionic currents were measured using excised inside-out patches to access the intracellular part of the channel. In the absence of Cd2þ, V476C concatemer mutants open and close normally. In the presence of Cd2þ, successive addition of V476C mutations to the subunits of the concatemer increasingly slowed down the closing of the channel at 120 mV. With all four V476C mutations, channels are locked open with Cd2þ.