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Precise Control of Temperature in Artificial Planar Lipid Bilayers to Modulate Different TRP Channels
250a
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.
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.