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Anesthetic-Sensitive Ion Channel Modulation Is Associated with a Molar Water Solubility Cut-Off Robert J

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Anesthetic-Sensitive Ion Channel Modulation Is Associated with a Molar Water Solubility Cut-Off Robert J Brosnan and Pham BMC Pharmacology and Toxicology (2018) 19:57 https://doi.org/10.1186/s40360-018-0244-z RESEARCH ARTICLE Open Access Anesthetic-sensitive ion channel modulation is associated with a molar water solubility cut-off Robert J. Brosnan* and Trung L. Pham Abstract Background: NMDA receptor modulation by hydrocarbons is associated with a molar water solubility cut-off. Low-affinity phenolic modulation of GABAA receptors is also associated with a cut-off, but at much lower molar solubility values. We hypothesized that other anesthetic-sensitive ion channels exhibit distinct cut-off effects associated with hydrocarbon molar water solubility, and that cut-off values are comparatively similar between related receptors than phylogenetically distant ones. Methods: Glycine or GABAA receptors or TREK-1, TRESK, Nav1.2, or Nav1.4 channels were expressed separately in frog oocytes. Two electrode voltage clamp techniques were used to study current responses in the presence and absence of hydrocarbon series from eight functional groups with progressively increasing size at saturated aqueous concentrations. Null response (cut-off) was defined by current measurements that were statistically indistinguishable between baseline and hydrocarbon exposure. Results: Ion channels exhibited cut-off effects associated with hydrocarbon molar water solubility in the following order of decreasing solubility: Nav1.2 ≈ Nav1.4 ≳ TRESK ≈ TREK-1 > GABAA >> glycine. Previously measured solubility cut-off values for NMDA receptors were intermediate between those for Nav1.4 and TRESK. Conclusions: Water solubility cut-off responses were present for all anesthetic-sensitive ion channels; distinct cut-off effects may exist for all cell surface receptors that are sensitive to volatile anesthetics. Suggested is the presence of amphipathic receptor sites normally occupied by water molecules that have dissociation constants inversely related to the cut-off solubility value. Poorly soluble hydrocarbons unable to reach concentrations sufficient to out-compete water for binding site access fail to modulate the receptor. Keywords: Anesthesia, Mechanism, Ion channel, Electrophysiology, Aliphatic Background ethers (with and without halogens), and various alkenes Protein-ligand interactions are commonly described using (with and without halogens); experimental inhaled anes- a “lock-and-key” model in which the protein and ligand thetics demonstrate even greater structural variety. Hence, have to fit together to have a chemical effect [1]. Although these drugs act as structurally diverse keys that each can inhaled anesthetics are presumed to exert their effects open the same lock. Moreover, a single inhaled anesthetic through ion channels and cell surface receptor proteins, can allosterically modulate function of a large number the diverse nature of both anesthetic ligands and the pro- of structurally diverse and phylogenetically unrelated teins they modulate challenge assumptions of a conserved ion channels—including many different ligand-gated ion complementary structure [2, 3]. Current and historical channels, voltage-gated ion channels, and leak channels— inhaled anesthetics include single atoms, triatomic mole- and cell surface receptors—including many different cules, various alkanes (with and without halogens), various channel-linked receptors, enzyme-linked receptors, and G protein-coupled receptors. Hence, these agents also * Correspondence: [email protected] act like a key that can open many different locks. Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Brosnan and Pham BMC Pharmacology and Toxicology (2018) 19:57 Page 2 of 13 Inhaled anesthetics typically have high median effective and were either administered morphine analgesia and re- concentrations consistent with low-affinity target interac- covered from anesthesia (first ovariectomy) or euthanized tions [4], and these agents can bind multiple amphipathic (second ovariectomy) by decapitation and double pithing sites on a protein of which at least some may be associated while anesthetized. The theca externa and mesovarium of with water molecules [5]. Anesthetic modulation of protein the removed ovary were disrupted manually, and oocytes function firstly depends upon the presence of an agent were defolliculated enzymatically by use of 0.2% collage- in sufficient concentration to desolvate, displace weakly- nase. Oocytes were stored in a modified Barth’s electrolyte bound water molecules within an allosteric binding site, solution until ready for use. This protocol was approved and only then bind to the allosteric site. We postulate that by the Institutional Animal Care and Use Committee at drugs with insufficient water solubility to competitively the University of California, Davis. displace water from this amphipathic allosteric site would be unable to bind and modulate the protein—a cut-off Receptor expression effect—even at a saturated aqueous phase drug concentra- Each anesthetic-sensitive receptor type was separately tion. These low-affinity amphipathic drug-receptor inter- expressed in different oocytes by injecting plasmids actions could be associated with different hydrocarbon containing sequences for receptor subunits which were solubility cut-off values for different proteins since the provided as gifts from the laboratory of R. Adron Harris water dissociation constants within critical allosteric sites (University of Texas, Austin). All plasmids were sequenced could also be different for different proteins. In support, and their identity confirmed by comparison to National N-methyl-d-aspartate (NMDA) receptor whole-cell currents Center for Biotechnology Information databases. Human are unchanged by saturated concentrations of organic voltage-gated Type II sodium channels (Nav1.2) were compounds once the hydrocarbon ω-end or ring size is expressed by intracytoplasmic injection of 5 ng RNA per increased such that the predicted molar water solubility oocyte containing the SCN2A gene; human voltage gated is less than approximately 1.1 mM; yet these same hydrocar- Type IV sodium channels (Nav1.4) were similarly expressed bons still are able to allosterically modulate γ-aminobutyric using RNA encoding the SCN4A gene. The human TRESK acid receptor type A (GABAA)currents[6]. However, channel (K2P18.1) was expressed by intracytoplasmic GABAA receptors may have their own hydrocarbon cut-off, injection of 5 ng RNA per oocyte containing the KCNK18 as GABAA receptor whole-cell currents are unchanged by a gene; the human TREK-1 channel (K2P2.1), was similarly series of substituted phenol and benzene rings once the expressed using RNA encoding the KCNK2 gene. Hetero- predicted molar water solubility of these compounds is trimeric GABAA receptors were expressed by a 1 ng oocyte less than 0.1 mM [7]. intranuclear co-injection of three different plasmids We hypothesize that cut-off effects associated with molar containing cytomegalovirus promoters and coding DNA water solubility is a generalizable feature of inhaled for one of thee subunits—human α1 (GABRA1), rat β2 anesthetic-sensitive ion channels and receptors. As a (GABRB2), or rat γ2s (GABRG2 short)—in a 1:1:10 ratio test, we evaluated responses of anesthetic-sensitive to ensure incorporation of the γ-subunit; this was channels and receptors to eight different functional confirmed by demonstrating receptor potentiation to groups of organic compounds, differing only by carbon 10 μM chlordiazepoxide during co-application with GABA. additions to the ω-endofachainortoaring,sothat Glycine receptors were expressed by intracytoplasmic we could study compounds of different molecular volumes injection of 5 ng RNA per oocyte containing the gene and carbon atoms but similar molar water solubility values for the human glycine α1 subunit (GLRA1). Nuclease- (and vice versa) and therefore distinguish between effects free water-injected oocytes served as negative controls caused by drug size versus solubility. Although inhaled for all experiments. anesthetics bind many different proteins, we studied six channels and receptors reported to contribute to immobil- Electrophysiology studies izing effects in vivo: voltage-gated sodium channels type II Oocytes were incubated at 18 °C for 1–4 days in fresh and type IV (Nav1.2 and Nav1.4), [8] TWIK-related spinal and filtered modified Barth’ssolutioncomposedof88mM cord channel (TRESK) [9], TWIK-related potassium NaCl, 1 mM KCl, 2.4 mM NaHCO3, 20 mM HEPES, channel type I (TREK-1) [10], GABAA receptors [11], 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, and glycine receptors [12]. 5 mM sodium pyruvate, gentamycin, penicillin, strepto- mycin, and corrected to pH = 7.4. All salts and antibiotics Methods were A.C.S. grade (Fisher Scientific, Pittsburgh, PA). Oocyte collection Oocytes were studied in a 250 μL linear-flow chamber
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