High‐affinity copper block of GABA<sub>A</sub> receptor‐mediated currents in acutely isolated cerebellar Purkinje cells of the rat

Шаронова, И. Н.; Vorobjev, Vladimir S.; Haas, Helmut L.

doi:10.1046/j.1460-9568.1998.00057.x

Cited by 52 publications

(49 citation statements)

References 23 publications

(29 reference statements)

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“…Effects of exogenous copper have been reported for several types of ion channels (73)(74)(75), but the binding sites are unknown. Recently, Li et al (76) reported effects of reducing agents on the Torpedo ClC-0 chloride channel, but exogenous copper (10 M) was without effect.…”

Section: Discussionmentioning

confidence: 99%

Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol

Liu¹,

Alexander

Serrano³

et al. 2006

Journal of Biological Chemistry

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In a previous study of T338C CFTR (cystic fibrosis transmembrane conductance regulator) we found that protons and thiol-directed reagents modified channel properties in a manner consistent with the hypothesis that this residue lies within the conduction path, but the observed reactivity was not consistent with the presence of a single thiolate species in the pore. Here we report results consistent with the notion that the thiol moiety can exist in at least three chemical states, the simple thiol, and two altered states. The technique of cysteine scanning mutagenesis has been widely used to probe the structure and functional properties of ion channels (1-8), including the cystic fibrosis transmembrane conductance regulator (CFTR) 4 (9 -11). The strategy is based on the notion that the reactive thiolate anion can interact with a variety of reagents including mixed disulfides (8,(12)(13)(14), alkylating reagents (12, 15, 16), and metals (17-20). In such experiments a typical first step is to screen various cysteine-substituted constructs for reactivity using, for example, reagents such as MTSET ϩ or MTSES Ϫ that react with the thiolate anion by means of thiol-disulfide exchange (14). The result is a pattern of reactivity, usually portrayed as the relative increase, decrease, or no change in conductance. In such an assay, if exposure to a reagent results in no change in conductance, it might be thought that the cysteine in question was not accessible to the reagents. It is recognized by practitioners, however, that a failure to respond could also reflect either the lack of any functional consequence of the reaction or altered reactivity of the engineered cysteine in its non-natural, protein environment. Although the latter possibility is generally recognized, potential mechanisms have not been studied in any detail (7,8,21). We report here evidence that a cysteine substituted into transmembrane segment 6 of CFTR can undergo spontaneous changes in its reactivity toward thiol-directed reagents that can produce conflicting results in a screening assay. These effects appear to be attributable to changes in the chemical state of the cysteine thiol. The goal of the experiments described here was to use thiol reactivity to operationally define different states of the cysteine at position 338 and to compare altered reactivity with that induced experimentally. The results are consistent with the hypothesis that the engineered cysteine is a component of an adventitious metal binding site that exhibits a high affinity for copper. MATERIALS AND METHODSMutagenesis and in Vitro Transcription-The methods used for mutagenesis and in vitro transcription were similar to those reported previously (11,22). Briefly, CFTR mutants were generated using the QuikChange TM site-directed mutagenesis kit from Stratagene. The sequences in the region of the mutation and in the entire PCR-generated region were confirmed by direct DNA sequencing.The T338C CFTR mutants used in this study were generated on two different CFTR backgrounds, either the ...

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Section: Discussionmentioning

confidence: 99%

Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol

Liu¹,

Alexander

Serrano³

et al. 2006

Journal of Biological Chemistry

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“…2 To whom correspondence should be addressed. (20). To exclude that the observed histamine effects depend on the complexation of such divalent cation contaminations, potentiation and dose-response curves experiments were performed in Ringer's solution prepared with ultrapure water (AMPUWA water, Fresenius, Bad Homburg, Germany) as suggested (20).…”

Section: Methodsmentioning

confidence: 99%

Histamine Action on Vertebrate GABAA Receptors

Saras¹,

Gisselmann²,

Vogt-Eisele

et al. 2008

Journal of Biological Chemistry

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Histamine is not only a crucial cytokine in the periphery but also an important neurotransmitter and neuromodulator in the brain. It is known to act on metabotropic H1-H4 receptors, but the existence of directly histamine-gated chloride channels in mammals has been suspected for many years. However, the molecular basis of such mammalian channels remained elusive, whereas in invertebrates, genes for histamine-gated channels have been already identified. In this report, we demonstrated that histamine can directly open vertebrate ion channels and identified ␤ subunits of GABA A receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric ␤ channels, histamine evoked currents with an EC 50 of 212 M (␤ 2 ) and 174 M (␤ 3 ), whereas GABA is only a very weak partial agonist. We tested several known agonists and antagonists for the histamine-binding site of H1-H4 receptors and described for ␤ channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of ␣ 1 ␤ 2 or ␣ 1 ␤ 2 ␥ 2 subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a ␥ 2 subunit-controlled manner. Despite the vast number of synthetic modulators of GABA A receptors widely used in medicine, which act on several distinct sites, only a few endogenous modulators have yet been identified. We show here for the first time that histamine modulates heteromultimeric GABA A receptors and may thus represent an endogenous ligand for an allosteric site.Ligand-gated ion channels mediate the fast responses of cells to neurotransmitters (1). A universal feature of ligand-gated ion channels subunits is a common topology, comprising four membrane-spanning segments (M1-M4) and a huge N-terminal extracellular domain with a hyperconserved cysteine loop motif. In vertebrates, this "Cys loop" family of phylogenetically related genes codes for anion and cation channels activated by acetylcholine and serotonin (cation channels) or GABA 3 and glycine (anion channels) (1, 2). Despite the many years of intensive research on such ion channels, recent reports revealed unexpected new findings about this channel family. In vertebrates, a gene for zinc-gated ion channels was recently discovered (3). In insects, new classes of ligand-gated chloride channels gated by histamine or pH and cation channels gated by GABA were reported (4 -7

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“…Cu 2ϩ inhibition of olfactory bulb neurons was similar to that of dorsal root ganglion neurons (Trombley and Shepherd, 1996). However, in cerebellar Purkinje cell GABA A receptor currents, maximal Cu 2ϩ block was only 60% and the IC 50 was lower (35 nM) (Sharonova et al, 1998) than in dorsal root ganglion neurons. Variations in Cu 2ϩ pharmacology may come from different region-specific GABA A receptor subunit subtype combinations.…”

Section: Discussionmentioning

confidence: 85%

“…However, several recent studies have shown that Cu 2ϩ inhibited GABA A receptor currents in native and transfected cells (Ma and Narahashi, 1993;Narahashi et al, 1994;Fisher and Mac-This study was supported by National Institutes of Health grant NS33300 (to R.L.M.). donald, 1998;Sharonova et al, 1998). In addition, Cu 2ϩ inhibited ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, N-methyl-D-aspartate, and glycine receptor currents (Trombley and Shepherd, 1996;Vlachova et al, 1996;Weiser and Wienrich, 1996) and blocked long-term potentiation in hippocampus (Doreulee et al, 1997).…”

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confidence: 99%

An N-Terminal Histidine Is the Primary Determinant of α Subunit-Dependent Cu²⁺Sensitivity of αβ3γ2L GABA_AReceptors

Kim

Macdonald²

2003

Mol Pharmacol

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Copper (Cu 2ϩ ) is a physiologically important cation and is released from nerve terminals. Cu 2ϩ modulates GABA A receptor currents in an ␣ subunit subtype-dependent manner; ␣1␤3␥2L receptors are more sensitive to Cu 2ϩ than ␣6␤3␥2L receptors. We compared the effect of Cu 2ϩ on ␣␤3␥2L receptors containing each of the six ␣ subtypes and generated ␣1/␣6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for ␣ subtype-dependent differences in Cu 2ϩ sensitivity. Whole-cell GABA A receptor currents were obtained from L929 fibroblasts coexpressing wildtype, chimeric and mutant ␣ subunits with ␤3 and ␥2L subunits. Maximal Cu 2ϩ inhibition of ␣1␤3␥2L and ␣2␤3␥2L receptor currents was larger (52.2 Ϯ 3.0 and 59.0 Ϯ 2.5%, respectively) than maximal inhibition of ␣3␤3␥2L, ␣4␤3␥2L, ␣5␤3␥2L, and ␣6␤3␥2L receptor currents (22.6 Ϯ 3.1, 19.2 Ϯ 3.4, 20.2 Ϯ 4.8, and 21.2 Ϯ 3.6%, respectively). Receptors containing chimeric constructs with ␣1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu 2ϩ to an extent similar to those with ␣1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu 2ϩ sensitivity. ␣1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the ␣2 subtype) conferred higher Cu 2ϩ sensitivity, and the H141 residue was the major determinant in the motif. The ␤3 subtype M2 domain residue H267, which is a major determinant of Zn 2ϩ inhibition, and ␣6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn 2ϩ sensitivity of the ␣6 subtype, also seemed to contribute to Cu 2ϩ inhibition. These data suggest that the Nterminal VR(Q)AECPMH motif in ␣1 and ␣2 subtypes is the major determinant of increased subtype-dependent inhibition by Cu 2ϩ , that residue H141 is the major determinant in that motif, and that Cu 2ϩ may also interact with GABA A receptors at sites similar to or overlapping Zn 2ϩ sites.Fast inhibitory synaptic transmission in the mammalian central nervous system is mediated primarily by GABA A receptors, which are members of a superfamily of ligandgated ion channels including nicotinic acetylcholine, glycine, and 5-hydroxytryptamine type 3 receptors. GABA A receptors are composed of a pentameric combination of subunits that form an intrinsic chloride ion channel. Each GABA A receptor subunit has a putative membrane topology consisting of a large N-terminal extracellular domain, four membrane spanning domains (TM1-TM4) and an extracellular C terminus.

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High‐affinity copper block of GABA_A receptor‐mediated currents in acutely isolated cerebellar Purkinje cells of the rat

Cited by 52 publications

References 23 publications

Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol

Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol

Histamine Action on Vertebrate GABAA Receptors

An N-Terminal Histidine Is the Primary Determinant of α Subunit-Dependent Cu²⁺Sensitivity of αβ3γ2L GABA_AReceptors

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High‐affinity copper block of GABAA receptor‐mediated currents in acutely isolated cerebellar Purkinje cells of the rat

Cited by 52 publications

References 23 publications

Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol

Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol

Histamine Action on Vertebrate GABAA Receptors

An N-Terminal Histidine Is the Primary Determinant of α Subunit-Dependent Cu2+Sensitivity of αβ3γ2L GABAAReceptors

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High‐affinity copper block of GABA_A receptor‐mediated currents in acutely isolated cerebellar Purkinje cells of the rat

An N-Terminal Histidine Is the Primary Determinant of α Subunit-Dependent Cu²⁺Sensitivity of αβ3γ2L GABA_AReceptors