Tandospirone-induced K+current in acutely dissociated rat dorsal raphe neurones (original) (raw)
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Experimental Brain Research, 2013
Voltage-gated potassium channels (Kv) Voltage-gated potassium channels regulate numerous and heterogeneous cell functions. In particular, they shorten action potentials duration, modulate the release of neurotransmitters, and control the excitability, electrical properties and firing pattern of neurons in the brain (Hille 2001). the first Kv channel was cloned from the Shaker mutant of Drosophila in 1987Drosophila in (tempel et al. 1987. Several other genes encoding for Kv channels have been identified afterward from many different species. Based on sequence relatedness, Kv channels have been classified in subfamilies by using the abbreviation Kvy.x (Chandy and Gutman 1993).
PLOS ONE, 2015
G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT 1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltageclamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT 1A autoreceptor-activated inwardly rectifying K + conductance in rat dorsal raphe 5-HT neurons. 5-HT 1A autoreceptoractivated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba 2+ (EC 50 = 9.4 μM, full block with 100 μM) and by SCH23390 (EC 50 = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT 1A autoreceptor-activated GIRK conductance with high potency (EC 50 = 33.6 nM), but incompletely, i.e.~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC 50 s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K + channels coupled to 5-HT 1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.
British Journal of Pharmacology, 1993
1 The biophysical and pharmacological properties of 5-hydroxytryptamine (5-HT)-evoked currents in rabbit nodose ganglion neurones in culture have been determined by use of the whole-cell and outside-out membrane patch recording modes of the patch-clamp technique. 2 In 49% of cells investigated the bath application of 10-5 M 5-HT at negative holding potentials elicited an inward current. The whole-cell response to 5-HT reversed in sign (E5-HT) at approximately -2 mV and exhibited inward rectification.
The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques
British Journal of Pharmacology, 1989
1 The characteristics of transmembrane currents evoked by 5-hydroxytryptamine (5-HT) in the neuroblastoma x Chinese hamster brain cell line and neuroblastoma clonal cell line NIE-115 have been studied under voltage-clamp conditions by the whole-cell recording and outside-out membrane patch modes of the patch-clamp technique. 2 In 73% of NCB-20 cells examined (n = 221), and all NIE-115 cells studied (n = 80), 5-HT (10pM) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5-HT reversed in sign at a potential of approximately -2 mV and demonstrated inward rectification. 3 The reversal potential of 5-HT-induced currents (ES-HT) recorded from either NCB-20 or NIE-115 cells was unaffected by total replacement of internal K+ by Cs'. In NIE-115 cells, reducing internal K+ concentration from 140 to 20mM produced a positive shift in E5-HT of approximately 28 mV, whereas reducing external Na' from 143 to 20mM was associated with a negative shift in ES-HT of about 37mV. A large reduction in internal Cl-concentration (from 144 to 6mM) had little effect on ES-HT. 4 5-HT-induced currents of NCB-20 cells were unaffected by methysergide (1 gM) or ketanserin (1pM), but were reversibly antagonized by GR38032F (0.1-1.OnM) with an IC50 of 0.25nm. GR 38032F (0.3nM) reduced 5-HT-induced currents in NIE-115 cells to approximately 26% of their control value. 5 On outside-out membrane patches excised from both NCB-20 and NlE-115 cells, 5-HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nM) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5-HT (1 M) in NlE-1 15 cells suggests that 5-HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside-out membrane patches to 5-HT cannot at present be resolved into clear single channel events.
Journal of Biological Chemistry, 2010
An inwardly rectifying K + current is present in atrial cardiac myocytes that is activated by acetylcholine (I KACh). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gβγ G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrialderived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbacholactivated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPγS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.
Regulation of heterologously expressed 5-HT1B receptors coupling to K channels in AtT-20 cells
British Journal of Pharmacology, 2018
BACKGROUND AND PURPOSE 5-HT 1B receptors are widely expressed GPCRs and a target of triptans, the most commonly prescribed anti-migraine drugs. There is very limited information about the acute, agonist-induced regulation of 5-HT 1B receptor signalling and so we sought to characterize this in a neuron-like system. EXPERIMENTAL APPROACH Epitope-tagged human 5-HT 1B receptors were expressed in mouse AtT20 cells. 5-HT 1B receptor signalling was assessed using whole-cell patch-clamp recordings of endogenous G protein-gated inwardly rectified potassium (GIRK) channels, and receptor localization measured using immunofluorescence. KEY RESULTS 5-HT (EC 50 65 nM) and sumatriptan (EC 50 165 nM) activated GIRK channels in AtT20 cells expressing 5-HT 1B receptors. Continuous application of both 5-HT (EC 50 120 nM) and sumatriptan (EC 50 280 nM) produced profound desensitization of 5-HT 1B receptor signalling within a few minutes. Complete recovery from desensitization was observed after 10 min. Both 5-HT and sumatriptan induced significant heterologous desensitization of SRIF (somatostatin)-activated GIRK currents, with the 5-HT-induced heterologous desensitization being blocked by the protein kinase inhibitor staurosporine. Both agonists induced modest 5-HT 1B receptor internalization, with a time course much slower than receptor desensitization. CONCLUSIONS AND IMPLICATIONS In AtT-20 cells, 5-HT 1B receptors undergo rapid and reversible desensitization at concentrations of agonist similar to those required to activate the receptor. Desensitization is incomplete, and the continued signalling of the receptor in the presence of the agonist may lead to cellular adaptations. Finally, 5-HT 1B receptor activation causes significant heterologous desensitization, which may lead to a reduced effectiveness of unrelated drugs in vivo.
The Journal of Physiology, 1999
1. G protein-regulated inward rectifier K¤ (GIRK) channels were over-expressed in dissociated rat superior cervical sympathetic (SCG) neurones by co-transfecting green fluorescent protein (GFP)-, GIRK1-and GIRK2-expressing plasmids using the biolistic technique. Membrane currents were subsequently recorded with whole-cell patch electrodes. 2. Co-transfected cells had larger Ba¥-sensitive inwardly rectifying currents and 13 mV more negative resting potentials (in 3 mÒ [K¤]o) than non-transfected cells, or cells transfected with GIRK1 or GIRK2 alone. 3. Carbachol (CCh, 1-30 ìÒ) increased the inwardly rectifying current in 70% of GIRK1+ GIRK2-transfected cells by 261 ± 53 % (n = 6, CCh 30 ìÒ) at −120 mV, but had no effect in non-transfected cells or in cells transfected with GIRK1 or GIRK2 alone. Pertussis toxin prevented the effect of carbachol but had no effect on basal currents. 4. The effect of CCh was antagonized by 6 nÒ tripitramine but not by 100 nÒ pirenzepine, consistent with activation of endogenous Mµ muscarinic acetylcholine receptors. 5. In contrast, inhibition of the voltage-activated Ca¥ current by CCh was antagonized by 100 nÒ pirenzepine but not by 6 nÒ tripitramine, indicating that it was mediated by MÚ muscarinic acetylcholine receptors. 6. We conclude that endogenous Mµ and MÚ muscarinic receptors selectively couple to GIRK currents and Ca¥ currents respectively, with negligible cross-talk. 8938
The Journal of General Physiology, 1988
A B ST RACT The role of a guanine nucleotide-binding protein (Gk) in the coupling between muscarinic receptor activation and opening of an inwardly rectifying K + channel [IK(M)] was examined in cardiac atrial myocytes, using hydrolysis-resistant GTP analogues. In the absence of muscarinic agonist, GTP analogues produced a membrane current characteristic of IK(M). The initial rate of appearance of this receptor-independent IK(M) was measured for the various analogues in order to explore the kinetic properties of IK(M) activation. We found that Ir~(M) activation is controlled solely by the intracellular analogue/GTP ratio and not by the absolute concentrations of the nucleotides. Analogues competed with GTP for binding to Gk with the following relative affinities: GTP'yS > GTP > GppNHp > GppCH2p. At sufficiently high intracellular concentrations, however, all GTP analogues produced the same rate of Is(M) activation. This analogue-independent limiting rate is likely to correspond to the rate of GDP release from inactive, GDP-bound Gk. Muscarinic receptor stimulation by nanomolar concentrations of acetylcholine (ACh), which do not elicit IS(M) under control conditions, catalyzed /V.(M) activation in the presence of GTP analogues. The rate of Gk activation by ACh (kAch) was found to be described by the simple relationship kAch = 8.4 X l0 s min-lM -~'[ACh] + 0.44 rain -~, the first term of which presumably reflects the agonist-catalyzed rate of GDP release from the Gk'GDP complex, while the second term corresponds to the basal rate of receptor-independent GDP release. Combined with the estimated K0.5 of the IK(M)-[ACh] dose-effect relationship, 160 nM, this result also allowed us to estimate the rate of Gk" GTP hydrolysis, k ..... ,to be near 135 min -j. These results provide, for the first time, a quantitative description of the salient features of G-protein function in vivo.
Electrophysiology of 5HT1A receptors in the rat hippocampus and cortex
Drug Development Research, 1992
Electrophysiological recordings from hippocampus and cortex have demonstrated that one of the most prominent effects of serotonin in these regions is a membrane hyperpolarization that effectively inhibits neuronal activity. The use of the in vitro brain slice preparation has allowed for detailed pharmacological and physiological studies of this response. Pharmacological analysis using agonists and antagonists indicates that these responses are mediated by activation of receptors of the 5-HT1A subtype. Buspirone, ipsapirone and 8-OHDPAT are all partial agonists at this receptor with 8-OHDPAT exhibiting an intrinsic activity approximately one-fourth that of serotonin. The ability of 5-HT1A receptor agonists to elicit a hyperpolarization is dependent on intracellular GTP, suggesting the involvement of a G protein in the transmembrane signalling mechanism. In agreement with this idea, injection of the stable GTP analog GTPγS renders the serotonin induced hyperpolarization irreversible, while GDPβS blunts its effects and pertussis toxin pretreatment blocks it. The 5-HT1A receptor induced hyperpolarization is mediated by an increase in potassium conductance. While the identity of the potassium channel remains to be determined, its basic characteristics identify it as belonging to a general class of inwardly rectifying G protein activated potassium channels ubiquitously distributed in neuronal and cardiac muscle tissues. In the rat hippocampus and cortex, most pyramidal cells co-express 5-HT1A with either 5-HT4 or 5-HT2 receptors, respectively, which in turn act to increase the ability of strong stimuli to excite these cells. As a result the net effect of serotonin on membrane excitability is dependent on the strength of incoming stimuli. Weak stimuli are depressed by the coactivation of these receptor subtypes while strong stimuli are enhanced. Thus the effects of selective 5-HT1A ligands are likely to depend not only on their direct effect on membrane excitability but also on how they alter ongoing serotonergic neurotransmission. © 1992 Wiley-Liss, Inc.
British Journal of Pharmacology, 1989
Belgium 1 Recently, we suggested that the D2-dopamine receptor involved in the inhibition of evoked [3H]-acetylcholine release from rat striatum is coupled to K+-channels. 2 In the present study, an attempt was made to elucidate further the role of these K+-channels, using the K+-channel blocking agents tetraethylammonium and 4-aminopyridine. With a superfusion method, the effects of both drugs on the D2-dopamine receptor-mediated inhibition of the electrically evoked release of [3H]-acetylcholine from rat striatal tissue slices was investigated. 3 Both tetraethylammonium (30 mM) and 4-aminopyridine (0.1 mM) significantly stimulated the electrically evoked release of [3H]-acetylcholine and completely abolished the effect of the selective D2-receptor agonist LY 171555 (1 gM) on evoked acetylcholine release. In addition, tetraethylammonium (0.03-30mM) and 4-aminopyridine (0.003-1 mM) strongly increased the basal (nonevoked) release of radioactivity in a concentration-dependent manner. The results suggest that the effect of the drugs on the basal release of radioactivity and on the electrically evoked release of acetylcholine cannot exclusively be explained by their action on K +-channels. 4 Furthermore, with the use of a receptor binding assay, data were obtained on the affinity of tetraethylammonium and 4-aminopyridine for D2-receptors and various other neurotransmitter recognition sites. At concentrations in which both drugs are known to block K+-channels, they were found to inhibit the specific binding of selective radioligands to their respective recognition sites. 5 It is concluded that due to their 'side-effects', both tetraethylammonium and 4-aminopyridine are of only limited value in the investigation of the alleged interaction between neurotransmitter receptors and K+-channels.