- Email: [email protected]
A Gain-of-Function TRPP2 Ion Channel Created by Mutating Single Amino Acid in the S5 Transmembrane Domain
Monday, February 13, 2017
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1223-Pos Board B291 Got Ion Accumulation or Depletion (In Your Recording)? Gilman E.S. Toombes, Mufeng Li, Shai D. Silberberg, Kenton J. Swartz. MPBU, NINDS, Bethesda, MD, USA. Although the flow of ions through channels or transporters can change ion concentrations at the membrane, the effects of ion depletion and accumulation are often neglected when designing and interpreting voltage and current clamp experiments. To address this issue, we implemented a simple, physically consistent 1-D convection-diffusion transport model that can be used to calculate time-dependent changes of voltage and ion concentrations. For cell-attached and excised-patch configurations, ion accumulation or depletion occurs on a millisecond timescale, although the exact rate can vary greatly depending on the pipette shape and position of the membrane patch. In contrast, in whole-cell and perforated-patch experiments the cytosol acts as a reservoir so ion concentrations change on a timescale of seconds with the rate determined primarily by the cell volume and pipette access resistance. For all patch-clamp configurations, steady-state ion accumulation or depletion depends on the electrode access resistance, rate of water transport and current carried by each and every ionic species. As a result, even when the net current is small and membrane voltage is effectively clamped, ion accumulation and depletion can give rise to time-dependent changes in current that resemble channel desensitization and/or dynamic ion selectivity. Simple guidelines to avoid these difficulties are proposed, as well as experimental tests to confirm that ion concentrations have been effectively clamped during a recording.
1226-Pos Board B294 The Isolated Voltage Sensing Domain of the Shaker Potassium Channel forms a Cation Channel Juan Zhao, Rikard Blunck. Physics and Physiology, University of Montreal, Montreal, QC, Canada. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). The energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the C-terminus proximal to S6. Energetic uncoupling between VSD and pore domain led to abolishment of ‘‘mode-shift’’ or ‘‘relaxation’’, a hysteresis in the voltage dependence of gating upon prolonged depolarization. However, relaxation was observed in the VSD of ciVSP which does not comprise a pore domain. Here, the isolated VSD of the Shaker-Kv channel (iVSD) construct was generated by truncating the channel C-terminal to S4 in the ShakerW434F-A359C background. The midpoint of the fluorescence voltage-relation (F-V) of iVSD was not significantly shifted compared to wildtype Shaker channel, but the voltage dependence was much shallower. The FV relations of Shaker-iVSD were continuously shifted to more hyperpolarized potentials at more depolarized holding potentials and did not saturate even at the extreme holding potentials, exhibiting a pronounced mode shift. Although Shaker-iVSD does not contain the ion conducting pore domain, it formed a cation-selective ion channel with a strong preference for protons. The ionic currents exhibited strong rectification with inward currents at hyperpolarized potentials at a holding potential of 0 mV. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain.
1224-Pos Board B292 Single Molecule FRET Imaging at Millimolar Concentrations in Zero Mode Waveguides David S. White1,2, Marcel P. Goldschen-Ohm1, Vadim A. Klenchin1, Randall H. Goldsmith2, Baron Chanda1. 1 Neuroscience, University of Wisconsin-Madsion, Madison, WI, USA, 2 Chemistry, University of Wisconsin-Madsion, Madison, WI, USA. Single-molecule fluorescence techniques are powerful tools which make feasible the elucidation of dynamic events underlying molecular mechanisms. Unfortunately, these techniques are often incompatible with physiological concentrations, as typical methods are unable to resolve single molecules beyond the high fluorescence background. Here, we combine zero-mode waveguide nanofabricated devices with single-molecule fluorescence energy transfer (smFRET) to resolve single-molecule binding dynamics at millimolar concentrations. As a model system, we report time-resolved single-molecule binding events of a fluorescently labeled cyclic nucleotide (fcGMP) to isolated cyclic-nucleotide binding domains (CNBD) from hyperpolarization-activated cyclic nucleotide-gated (HCN) channels at up to a millimolar concentration. This technique extends the resolution of single-molecules to ~1,000-fold higher concentrations, thus enabling the study of the weak binding interactions that govern a broad spectrum of biological processes.
1227-Pos Board B295 HCN Channel Gating Studied with tmFRET and a Fluorescent Noncanonical Amino Acid Teresa K. Aman, William N. Zagotta. Physiology and Biophysics, University of Washington, Seattle, WA, USA. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are members of the voltage-gated channel family, activated by hyperpolarizing voltages, and modulated by direct binding of cyclic nucleotides like cAMP. HCN channels are tetramers, with each subunit containing six transmembrane segments (S1S6), followed by a C-linker region that connects the cyclic nucleotide-binding domain (CNBD) to the channel pore. We are studying rearrangements in the C-linker region upon cyclic nucleotide binding and channel activation in the sea urchin HCN channel using transition metal ion FRET (tmFRET). Specifically, we incorporated a fluorescent noncanonical amino acid (Anap) into the channel as a FRET donor, and a metal binding site for a FRET acceptor. Two preparations were used for measuring Anap fluorescence: unroofed plasma membrane sheets and inside-out patches from HEK-293T/17 cells. Anap was incorporated into the S4-S5 linker of each channel subunit (F359Anap and W355Anap). To explore the movement of the A’ helix directly following the S6 segment with respect to Anap in the S4-S5 linker, we added a dihistidine motif to bind metal, and applied the metal ion cobalt (Co2þ). Because Anap emission overlaps with Co2þ absorption but Co2þ does not fluoresce, tmFRET is observed as Anap quenching. In unroofed cells, the quenching of F359Anap was 7.4% greater with a histidine pair in the A’ helix compared to the no histidine control. Interestingly, this difference was doubled in the presence of cAMP. Using the ˚ closer Fo¨rster equation, we estimate that cAMP caused the A’ helix to move ~3 A to the S4-S5 linker. In preliminary inside-out patch-clamp fluorometry experiments, F359Anap quenching with 10 mM Co2þ was similar compared to unroofed cells, and did not vary with negative voltage steps. These results indicate that the A’ helix plays an important role in translating cyclic nucleotide binding in the CNBD to modulating channel gating at the pore.
1225-Pos Board B293 Biophysical Characterization of ASAP1 Elizabeth E.L. Lee, Francisco Bezanilla. University of Chicago, Chicago, IL, USA. Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. ASAP1 is based on the Gallus gallus voltage sensitive phosphatase (GgVSP) with a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Expressing this molecule in Xenopus oocytes and recording fluorescence simultaneously with gating current using the cutopen voltage clamp technique, our studies show that the gating currents of ASAP1 are significantly faster than CiVSP. In ASAP1, gating current kinetics and fluorescence kinetics track each other closely. Interestingly, we found that while ASAP1 has a split QV curve, the removal of the GFP from the S3-S4, transforming it back to GgVSP, eliminates the split in the QV curve. Moreover, ASAP1 has a significantly shorter lag between gating and fluorescence (<0.5ms) than Arclight (~10ms). We have attempted to improve ASAP1 as optical reporter of membrane potential, by changing the characteristics of the voltage sensor, as revealed by gating currents and fluorescence. V160S is homologous to the mutation V363T in Shaker which accelerates the rate of activation. The kinetics of V160S fluorescence are slightly accelerated and more closely track gating kinetics, although it has a slight decrease in aF/F. Q153 functions much as R217 in CiVSP, by tuning the QV curve where changing residue 153 shifts the QV curve. These studies indicate that ASAP1 has QV and gating kinetics that can be modified to further improve its fluorescence response to voltage. Support NIH: GM030376.
TRP Channels II 1228-Pos Board B296 A Gain-of-Function TRPP2 Ion Channel Created by Mutating Single Amino Acid in the S5 Transmembrane Domain Mahmud Arif Pavel1, Caixia Lv2, Courtney Ng1, Parul Kashyap1, Clarissa Lam1, Victoria Valentino1, Helen Fung1, Thomas Campbell1, David Zenisek2, Nathalia G. Holtzman3, Yong Yu1. 1 Biological Sciences, St. John’s University, Queens, NY, USA, 2Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA, 3Department of Biology, Queens College, City University of New York, Queens, NY, USA. Mutations in polycystin-1 and TRPP2 (transient receptor potential polycystin 2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as cation channel when it is homomeric assembled, and when it forms a complex with polycystin-1. Studies on the function and regulation of
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Monday, February 13, 2017
TRPP2 are significantly delayed due to the lack of knowledge on its activation mechanism. Here, by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, we generated the constitutively active gain-offunction (GOF) mutant TRPP2_F604P and studied functional properties of this GOF channel. With this mutant channel, we identified D643, a negatively charged amino acid in the pore which plays a crucial role in determining channel permeability of TRPP2. We also found that extracellular divalent ions, including Ca2þ, inhibit the permeation of monovalent ions by blocking the TRPP2 channel pore. On the GOF background, the effects of nine ADPKD pathogenic mutations on channel function were tested, and we concluded that some pathogenic mutations have intact TRPP2 channel activity. We further investigated the in vivo function of the GOF TRPP2 in zebrafish embryos. Compared to wild-type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, caused by down-regulation of endogenous TRPP2 expression. Thus, we showed that the GOF TRPP2 channel could serve as a powerful tool in the TRPP2 study. The GOF channel may also have potential use in clinical applications. 1229-Pos Board B297 Precise Control of Temperature in Artificial Planar Lipid Bilayers to Modulate Different TRP Channels Conrad Weichbrodt1, Jiajun Wang1,2, Mohamed Kreir3, Matthias Beckler1, Alison Obergrussberger1, Ilka Rinke1, Michael George1, Sonja Stoelze-Feix1, Andrea Br€ uggemann1, Niels Fertig1. 1 Nanion Technologies GmbH, Munich, Germany, 2Jacobs University, Bremen, Germany, 3Discovery Sciences, Preclinical Development & Safety, Janssen Pharmaceutical Research & Development, Beerse, Belgium. Thermal Transient Receptor Potential (TRP) channels belong to the large family of TRP channels. They are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems. They have been shown to be directly activated by heat or cold in physiologically relevant temperature ranges, but are also activated by mechanostimulation and various ligands. Understanding the mechanisms of temperature activation could lead to the discovery of novel compounds with differing effects on ligand activation and temperature activation for the treatment of pain and other disease states, with fewer side effects. We have employed parallel planar lipid bilayer instrumentation (Orbit mini, Nanion) to study different reconstituted thermo-TRP channels, particularly the purified human TRP-A1, -V1, -V3 and -M8 channels. Planar lipid bilayers can be formed in the Orbit family systems by painting lipids in organic solvents over Micro Electrode Cavity Array (MECA) chips, a 2 by 2 array of circular micro-cavities in a highly inert polymer. The reconstitution of TRP channels is achieved by adding the purified proteins directly to the bilayers. In this study, we demonstrate the activation of the different TRP channels by cold and heat in a fully controlled environment using artificial membranes together with a Peltier element integrated in the Orbit mini setup to actively cool and heat the system to the desired temperatures (51 C) via an optional temperature control system. Furthermore, we compared our Q10 data from purified proteins to TRP channels expressed in HEK cells (Millipore, Chantest) using an automated patch clamp platform (Patchliner, Nanion) with temperature control. 1230-Pos Board B298 Assessment of Endogenous and Exogenous Modulators of the TRPM7 Channel in Planar Lipid Bilayers Lusine Demirkhanyan1, Tyler Dawson1, Thomas Gudermann2, Vladimir Chubanov2, Eleonora Zakharian1. 1 Department of Cancer Biology and Pharmacology, The University of Illinois at Chicago, Peoria, IL, USA, 2Walther-Straub Institute of Pharmacology and Toxicology, LMU Munich, Munich, Germany. TRPM7 is ubiquitously expressed divalent cation-selective ion channel. The channel subunit contains the alpha-kinase domain, which is located at the C-terminus. TRPM7 channels are implicated in a variety of cellular processes, including cell proliferation, differentiation, survival, death, Mg2þ homeostasis, and oxidative stress. TRPM7 is essential for the early embryonic development as the knockout of its gene results in embryonic lethality of mice. Furthermore, TRPM7 plays important roles in thymopoiesis, kidney morphogenesis, cardiac rhythmicity and immune responses. Several endogenous and exogenous modulators of TRPM7 channel have been suggested. However, molecular mechanisms of their action on the TRPM7 channel remain poorly understood. In this work, we aimed the incorporation and functional characterization of the purified TRPM7 channel in the defined reconstituted system. The mouse TRPM7 channel protein was purified by immunoprecipitation from HEK-293 cells transiently expressing the protein. The channel was then incorporated into planar lipid bilayers, consisting of a mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolaminein
(POPE) at 3:1 ratio. We found that TRPM7 activity is directly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2) and Mg2þ. In the presence of 2.5 mM PIP2, the TRPM7 channel exhibited highly organized burst opening, confirming the requirement of the phosphoinositide previously observed in the cellular system. An addition of the TRPM7 agonist, naltriben, (2.5 mM) further increased the open probability, thus enhancing the channel activity. In the bilayers, TRPM7 exhibited a strong outward rectification reflected both at the single channel conductance level and open probability. The mean slope conductance of an outward current was in the range of ~38.4 pS, whereas an inward current conductance was ~ 18.8 pS. In the presence of PIP2 alone, the channel displayed sustained low open probability (Po< 0.1) at all voltages of the inward current, and a voltage-dependent increase in Po of the outward current (~0.3 at 100 mV). Upon application of naltriben, Po of inwardly oriented openings essentially remained unchanged, while at outward Po almost doubled (~0.6 at 100 mV). Effects of naltriben- and PIP2-induced activities were abolished by the TRPM7 antagonist, NS8593, (1 mM), suggesting that TRPM7 directly interacts with PIP2, naltriben, and NS8593 in a voltage-dependent manner. To summarize, this study demonstrates a successful incorporation of the active TRPM7 channel in the bilayer system allowing the elucidation of mechanisms related to the channel opening and effects of endogenous and synthetic regulators. 1231-Pos Board B299 Effects of Ginger and its Pungent Constituents on Transient Receptor Potential Channels Young-Soo Kim1, Chan Sik Hong2, Sang Weon Lee1, Joo Hyun Nam3, Byung Joo Kim4. 1 Department of Neurosurgery, College of Medicine, Pusan National University, Yangsan Hospital, Yangsan, Korea, Republic of, 2College of Medicine, Chosun University, Gwangjoo, Korea, Republic of, 3College of Medicine, Dongguk University, Kyungjoo, Korea, Republic of, 4School of Korean Medicine, Pusan National University, Yangsan, Korea, Republic of. Ginger extract is used as an analeptic in herbal medicine and has been reported to exhibit antioxidant effects. TRPC5 (transient receptor potential canonical 5), TRPM7 (melastatin 7), and TRPA1 (ankyrin 1) are nonselective cation channels that are modulated by reactive oxygen/nitrogen species (ROS/RNS) and subsequently control various cellular processes. The aim of this study was to evaluate whether ginger and its pungent constituents modulate these channels exhibit antioxidant effects. It was found TRPC5 and TRPA1 currents were modulated by ginger extract and by its pungent constituents, [6]-gingerols, zingerone, and [6]-shogaol. In particular, [6]-shogaol strongly and dosedependently inhibited TRPC5 currents with an IC50 of ~18.3 mM. Furthermore, the strong dose-dependent activation of TRPA1 currents by [6]-shogaol was abolished by A-967079 (a selective TRPA1 inhibitor). However, ginger extract and its pungent constituents had no effect on TRPM7 currents. These results suggest the antioxidative effects of ginger extract and its pungent constituents involve TRPC5 and TRPA1 and that [6]-shogaol is predominantly responsible for the regulation of TRPC5 and TRPA1 currents by ginger extract. 1232-Pos Board B300 Modulation by Phenolic Compounds Provides Novel Insight into the Mechanisms of TRPA1 Activation Justyna B. Startek, Andrei Segal, Thomas Voets, Karel Talavera. Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. Transient receptor potential ankyrin 1 (TRPA1) is a cation channel activated by a large number of noxious chemicals found in many plants, foods, cosmetics, drugs and pollutants. These substances belong to different chemical classes with the specific functional groups responsible for the channel activation. Numerous TRPA1 agonists are highly reactive electrophiles, and have ability to bind and modify thiol groups in the cytosolic part of the channel. Another group of compounds contains a large number of non-electrophiles, such as menthol, clotrimazole and nicotine, which are unable to covalently modify TRPA1. Non-electrophilic agonists have extremely different chemical structures and functions, so it seems very unlikely that a single channel structure or binding pocket could accommodate direct binding of so many diverse compounds. However, one common feature of many of these species is the ability to partition into the plasma membrane, which may in turn induce mechanical perturbations. Using intracellular calcium measurements and patch-clamp recordings we found that non-electrophilic phenol derivatives activate mouse TRPA1 and that this effect is prevented by the specific channel inhibitor HC030031. The analysis of the effects of different phenol derivatives revealed a reduction of the EC50 for TRPA1 activation with increasing length of carbon side chain in positions orto, meta and para on the phenol ring. The lengthening of the carbonyl side chain correlates also with the increase in octanol/water partition coefficient and the ability of the compounds to insert into cellular membranes.
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1223-Pos Board B291 Got Ion Accumulation or Depletion (In Your Recording)? Gilman E.S. Toombes, Mufeng Li, Shai D. Silberberg, Kenton J. Swartz. MPBU, NINDS, Bethesda, MD, USA. Although the flow of ions through channels or transporters can change ion concentrations at the membrane, the effects of ion depletion and accumulation are often neglected when designing and interpreting voltage and current clamp experiments. To address this issue, we implemented a simple, physically consistent 1-D convection-diffusion transport model that can be used to calculate time-dependent changes of voltage and ion concentrations. For cell-attached and excised-patch configurations, ion accumulation or depletion occurs on a millisecond timescale, although the exact rate can vary greatly depending on the pipette shape and position of the membrane patch. In contrast, in whole-cell and perforated-patch experiments the cytosol acts as a reservoir so ion concentrations change on a timescale of seconds with the rate determined primarily by the cell volume and pipette access resistance. For all patch-clamp configurations, steady-state ion accumulation or depletion depends on the electrode access resistance, rate of water transport and current carried by each and every ionic species. As a result, even when the net current is small and membrane voltage is effectively clamped, ion accumulation and depletion can give rise to time-dependent changes in current that resemble channel desensitization and/or dynamic ion selectivity. Simple guidelines to avoid these difficulties are proposed, as well as experimental tests to confirm that ion concentrations have been effectively clamped during a recording.
1226-Pos Board B294 The Isolated Voltage Sensing Domain of the Shaker Potassium Channel forms a Cation Channel Juan Zhao, Rikard Blunck. Physics and Physiology, University of Montreal, Montreal, QC, Canada. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). The energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the C-terminus proximal to S6. Energetic uncoupling between VSD and pore domain led to abolishment of ‘‘mode-shift’’ or ‘‘relaxation’’, a hysteresis in the voltage dependence of gating upon prolonged depolarization. However, relaxation was observed in the VSD of ciVSP which does not comprise a pore domain. Here, the isolated VSD of the Shaker-Kv channel (iVSD) construct was generated by truncating the channel C-terminal to S4 in the ShakerW434F-A359C background. The midpoint of the fluorescence voltage-relation (F-V) of iVSD was not significantly shifted compared to wildtype Shaker channel, but the voltage dependence was much shallower. The FV relations of Shaker-iVSD were continuously shifted to more hyperpolarized potentials at more depolarized holding potentials and did not saturate even at the extreme holding potentials, exhibiting a pronounced mode shift. Although Shaker-iVSD does not contain the ion conducting pore domain, it formed a cation-selective ion channel with a strong preference for protons. The ionic currents exhibited strong rectification with inward currents at hyperpolarized potentials at a holding potential of 0 mV. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain.
1224-Pos Board B292 Single Molecule FRET Imaging at Millimolar Concentrations in Zero Mode Waveguides David S. White1,2, Marcel P. Goldschen-Ohm1, Vadim A. Klenchin1, Randall H. Goldsmith2, Baron Chanda1. 1 Neuroscience, University of Wisconsin-Madsion, Madison, WI, USA, 2 Chemistry, University of Wisconsin-Madsion, Madison, WI, USA. Single-molecule fluorescence techniques are powerful tools which make feasible the elucidation of dynamic events underlying molecular mechanisms. Unfortunately, these techniques are often incompatible with physiological concentrations, as typical methods are unable to resolve single molecules beyond the high fluorescence background. Here, we combine zero-mode waveguide nanofabricated devices with single-molecule fluorescence energy transfer (smFRET) to resolve single-molecule binding dynamics at millimolar concentrations. As a model system, we report time-resolved single-molecule binding events of a fluorescently labeled cyclic nucleotide (fcGMP) to isolated cyclic-nucleotide binding domains (CNBD) from hyperpolarization-activated cyclic nucleotide-gated (HCN) channels at up to a millimolar concentration. This technique extends the resolution of single-molecules to ~1,000-fold higher concentrations, thus enabling the study of the weak binding interactions that govern a broad spectrum of biological processes.
1227-Pos Board B295 HCN Channel Gating Studied with tmFRET and a Fluorescent Noncanonical Amino Acid Teresa K. Aman, William N. Zagotta. Physiology and Biophysics, University of Washington, Seattle, WA, USA. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are members of the voltage-gated channel family, activated by hyperpolarizing voltages, and modulated by direct binding of cyclic nucleotides like cAMP. HCN channels are tetramers, with each subunit containing six transmembrane segments (S1S6), followed by a C-linker region that connects the cyclic nucleotide-binding domain (CNBD) to the channel pore. We are studying rearrangements in the C-linker region upon cyclic nucleotide binding and channel activation in the sea urchin HCN channel using transition metal ion FRET (tmFRET). Specifically, we incorporated a fluorescent noncanonical amino acid (Anap) into the channel as a FRET donor, and a metal binding site for a FRET acceptor. Two preparations were used for measuring Anap fluorescence: unroofed plasma membrane sheets and inside-out patches from HEK-293T/17 cells. Anap was incorporated into the S4-S5 linker of each channel subunit (F359Anap and W355Anap). To explore the movement of the A’ helix directly following the S6 segment with respect to Anap in the S4-S5 linker, we added a dihistidine motif to bind metal, and applied the metal ion cobalt (Co2þ). Because Anap emission overlaps with Co2þ absorption but Co2þ does not fluoresce, tmFRET is observed as Anap quenching. In unroofed cells, the quenching of F359Anap was 7.4% greater with a histidine pair in the A’ helix compared to the no histidine control. Interestingly, this difference was doubled in the presence of cAMP. Using the ˚ closer Fo¨rster equation, we estimate that cAMP caused the A’ helix to move ~3 A to the S4-S5 linker. In preliminary inside-out patch-clamp fluorometry experiments, F359Anap quenching with 10 mM Co2þ was similar compared to unroofed cells, and did not vary with negative voltage steps. These results indicate that the A’ helix plays an important role in translating cyclic nucleotide binding in the CNBD to modulating channel gating at the pore.
1225-Pos Board B293 Biophysical Characterization of ASAP1 Elizabeth E.L. Lee, Francisco Bezanilla. University of Chicago, Chicago, IL, USA. Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. ASAP1 is based on the Gallus gallus voltage sensitive phosphatase (GgVSP) with a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Expressing this molecule in Xenopus oocytes and recording fluorescence simultaneously with gating current using the cutopen voltage clamp technique, our studies show that the gating currents of ASAP1 are significantly faster than CiVSP. In ASAP1, gating current kinetics and fluorescence kinetics track each other closely. Interestingly, we found that while ASAP1 has a split QV curve, the removal of the GFP from the S3-S4, transforming it back to GgVSP, eliminates the split in the QV curve. Moreover, ASAP1 has a significantly shorter lag between gating and fluorescence (<0.5ms) than Arclight (~10ms). We have attempted to improve ASAP1 as optical reporter of membrane potential, by changing the characteristics of the voltage sensor, as revealed by gating currents and fluorescence. V160S is homologous to the mutation V363T in Shaker which accelerates the rate of activation. The kinetics of V160S fluorescence are slightly accelerated and more closely track gating kinetics, although it has a slight decrease in aF/F. Q153 functions much as R217 in CiVSP, by tuning the QV curve where changing residue 153 shifts the QV curve. These studies indicate that ASAP1 has QV and gating kinetics that can be modified to further improve its fluorescence response to voltage. Support NIH: GM030376.
TRP Channels II 1228-Pos Board B296 A Gain-of-Function TRPP2 Ion Channel Created by Mutating Single Amino Acid in the S5 Transmembrane Domain Mahmud Arif Pavel1, Caixia Lv2, Courtney Ng1, Parul Kashyap1, Clarissa Lam1, Victoria Valentino1, Helen Fung1, Thomas Campbell1, David Zenisek2, Nathalia G. Holtzman3, Yong Yu1. 1 Biological Sciences, St. John’s University, Queens, NY, USA, 2Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA, 3Department of Biology, Queens College, City University of New York, Queens, NY, USA. Mutations in polycystin-1 and TRPP2 (transient receptor potential polycystin 2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as cation channel when it is homomeric assembled, and when it forms a complex with polycystin-1. Studies on the function and regulation of
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Monday, February 13, 2017
TRPP2 are significantly delayed due to the lack of knowledge on its activation mechanism. Here, by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, we generated the constitutively active gain-offunction (GOF) mutant TRPP2_F604P and studied functional properties of this GOF channel. With this mutant channel, we identified D643, a negatively charged amino acid in the pore which plays a crucial role in determining channel permeability of TRPP2. We also found that extracellular divalent ions, including Ca2þ, inhibit the permeation of monovalent ions by blocking the TRPP2 channel pore. On the GOF background, the effects of nine ADPKD pathogenic mutations on channel function were tested, and we concluded that some pathogenic mutations have intact TRPP2 channel activity. We further investigated the in vivo function of the GOF TRPP2 in zebrafish embryos. Compared to wild-type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, caused by down-regulation of endogenous TRPP2 expression. Thus, we showed that the GOF TRPP2 channel could serve as a powerful tool in the TRPP2 study. The GOF channel may also have potential use in clinical applications. 1229-Pos Board B297 Precise Control of Temperature in Artificial Planar Lipid Bilayers to Modulate Different TRP Channels Conrad Weichbrodt1, Jiajun Wang1,2, Mohamed Kreir3, Matthias Beckler1, Alison Obergrussberger1, Ilka Rinke1, Michael George1, Sonja Stoelze-Feix1, Andrea Br€ uggemann1, Niels Fertig1. 1 Nanion Technologies GmbH, Munich, Germany, 2Jacobs University, Bremen, Germany, 3Discovery Sciences, Preclinical Development & Safety, Janssen Pharmaceutical Research & Development, Beerse, Belgium. Thermal Transient Receptor Potential (TRP) channels belong to the large family of TRP channels. They are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems. They have been shown to be directly activated by heat or cold in physiologically relevant temperature ranges, but are also activated by mechanostimulation and various ligands. Understanding the mechanisms of temperature activation could lead to the discovery of novel compounds with differing effects on ligand activation and temperature activation for the treatment of pain and other disease states, with fewer side effects. We have employed parallel planar lipid bilayer instrumentation (Orbit mini, Nanion) to study different reconstituted thermo-TRP channels, particularly the purified human TRP-A1, -V1, -V3 and -M8 channels. Planar lipid bilayers can be formed in the Orbit family systems by painting lipids in organic solvents over Micro Electrode Cavity Array (MECA) chips, a 2 by 2 array of circular micro-cavities in a highly inert polymer. The reconstitution of TRP channels is achieved by adding the purified proteins directly to the bilayers. In this study, we demonstrate the activation of the different TRP channels by cold and heat in a fully controlled environment using artificial membranes together with a Peltier element integrated in the Orbit mini setup to actively cool and heat the system to the desired temperatures (51 C) via an optional temperature control system. Furthermore, we compared our Q10 data from purified proteins to TRP channels expressed in HEK cells (Millipore, Chantest) using an automated patch clamp platform (Patchliner, Nanion) with temperature control. 1230-Pos Board B298 Assessment of Endogenous and Exogenous Modulators of the TRPM7 Channel in Planar Lipid Bilayers Lusine Demirkhanyan1, Tyler Dawson1, Thomas Gudermann2, Vladimir Chubanov2, Eleonora Zakharian1. 1 Department of Cancer Biology and Pharmacology, The University of Illinois at Chicago, Peoria, IL, USA, 2Walther-Straub Institute of Pharmacology and Toxicology, LMU Munich, Munich, Germany. TRPM7 is ubiquitously expressed divalent cation-selective ion channel. The channel subunit contains the alpha-kinase domain, which is located at the C-terminus. TRPM7 channels are implicated in a variety of cellular processes, including cell proliferation, differentiation, survival, death, Mg2þ homeostasis, and oxidative stress. TRPM7 is essential for the early embryonic development as the knockout of its gene results in embryonic lethality of mice. Furthermore, TRPM7 plays important roles in thymopoiesis, kidney morphogenesis, cardiac rhythmicity and immune responses. Several endogenous and exogenous modulators of TRPM7 channel have been suggested. However, molecular mechanisms of their action on the TRPM7 channel remain poorly understood. In this work, we aimed the incorporation and functional characterization of the purified TRPM7 channel in the defined reconstituted system. The mouse TRPM7 channel protein was purified by immunoprecipitation from HEK-293 cells transiently expressing the protein. The channel was then incorporated into planar lipid bilayers, consisting of a mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolaminein
(POPE) at 3:1 ratio. We found that TRPM7 activity is directly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2) and Mg2þ. In the presence of 2.5 mM PIP2, the TRPM7 channel exhibited highly organized burst opening, confirming the requirement of the phosphoinositide previously observed in the cellular system. An addition of the TRPM7 agonist, naltriben, (2.5 mM) further increased the open probability, thus enhancing the channel activity. In the bilayers, TRPM7 exhibited a strong outward rectification reflected both at the single channel conductance level and open probability. The mean slope conductance of an outward current was in the range of ~38.4 pS, whereas an inward current conductance was ~ 18.8 pS. In the presence of PIP2 alone, the channel displayed sustained low open probability (Po< 0.1) at all voltages of the inward current, and a voltage-dependent increase in Po of the outward current (~0.3 at 100 mV). Upon application of naltriben, Po of inwardly oriented openings essentially remained unchanged, while at outward Po almost doubled (~0.6 at 100 mV). Effects of naltriben- and PIP2-induced activities were abolished by the TRPM7 antagonist, NS8593, (1 mM), suggesting that TRPM7 directly interacts with PIP2, naltriben, and NS8593 in a voltage-dependent manner. To summarize, this study demonstrates a successful incorporation of the active TRPM7 channel in the bilayer system allowing the elucidation of mechanisms related to the channel opening and effects of endogenous and synthetic regulators. 1231-Pos Board B299 Effects of Ginger and its Pungent Constituents on Transient Receptor Potential Channels Young-Soo Kim1, Chan Sik Hong2, Sang Weon Lee1, Joo Hyun Nam3, Byung Joo Kim4. 1 Department of Neurosurgery, College of Medicine, Pusan National University, Yangsan Hospital, Yangsan, Korea, Republic of, 2College of Medicine, Chosun University, Gwangjoo, Korea, Republic of, 3College of Medicine, Dongguk University, Kyungjoo, Korea, Republic of, 4School of Korean Medicine, Pusan National University, Yangsan, Korea, Republic of. Ginger extract is used as an analeptic in herbal medicine and has been reported to exhibit antioxidant effects. TRPC5 (transient receptor potential canonical 5), TRPM7 (melastatin 7), and TRPA1 (ankyrin 1) are nonselective cation channels that are modulated by reactive oxygen/nitrogen species (ROS/RNS) and subsequently control various cellular processes. The aim of this study was to evaluate whether ginger and its pungent constituents modulate these channels exhibit antioxidant effects. It was found TRPC5 and TRPA1 currents were modulated by ginger extract and by its pungent constituents, [6]-gingerols, zingerone, and [6]-shogaol. In particular, [6]-shogaol strongly and dosedependently inhibited TRPC5 currents with an IC50 of ~18.3 mM. Furthermore, the strong dose-dependent activation of TRPA1 currents by [6]-shogaol was abolished by A-967079 (a selective TRPA1 inhibitor). However, ginger extract and its pungent constituents had no effect on TRPM7 currents. These results suggest the antioxidative effects of ginger extract and its pungent constituents involve TRPC5 and TRPA1 and that [6]-shogaol is predominantly responsible for the regulation of TRPC5 and TRPA1 currents by ginger extract. 1232-Pos Board B300 Modulation by Phenolic Compounds Provides Novel Insight into the Mechanisms of TRPA1 Activation Justyna B. Startek, Andrei Segal, Thomas Voets, Karel Talavera. Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. Transient receptor potential ankyrin 1 (TRPA1) is a cation channel activated by a large number of noxious chemicals found in many plants, foods, cosmetics, drugs and pollutants. These substances belong to different chemical classes with the specific functional groups responsible for the channel activation. Numerous TRPA1 agonists are highly reactive electrophiles, and have ability to bind and modify thiol groups in the cytosolic part of the channel. Another group of compounds contains a large number of non-electrophiles, such as menthol, clotrimazole and nicotine, which are unable to covalently modify TRPA1. Non-electrophilic agonists have extremely different chemical structures and functions, so it seems very unlikely that a single channel structure or binding pocket could accommodate direct binding of so many diverse compounds. However, one common feature of many of these species is the ability to partition into the plasma membrane, which may in turn induce mechanical perturbations. Using intracellular calcium measurements and patch-clamp recordings we found that non-electrophilic phenol derivatives activate mouse TRPA1 and that this effect is prevented by the specific channel inhibitor HC030031. The analysis of the effects of different phenol derivatives revealed a reduction of the EC50 for TRPA1 activation with increasing length of carbon side chain in positions orto, meta and para on the phenol ring. The lengthening of the carbonyl side chain correlates also with the increase in octanol/water partition coefficient and the ability of the compounds to insert into cellular membranes.