M4 muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT20 cells

doi:10.1016/0304-3940(92)90576-S

Neuroscience Letters

Volume 147, Issue 2, 7 December 1992, Pages 125-130

https://doi.org/10.1016/0304-3940(92)90576-S Get rights and content

Abstract

The modulation of an inwardly rectifying potassium conductance by muscarinic receptor stimulation was studied in the AtT-20 pituitary cell line, using the whole-cell patch-clamp technique. Only m4 mRNA was detected in these cells, thus, it is assumed that the actions of muscarinic receptor stimulation are mediated by the m4 receptor. AtT-20 cells express a slowly activating inwardly rectifying potassium conductance. Application of acetylcholine (ACh), resulted in an atropine sensitive, reversible increase in inwardly rectifying current. The ACh-induced current differed from the current recorded in control, in that it was fast activating, while the control current was slowly activating. Inclusion of GTPγS in the patch pipette activated an inward current with characteristics similar to the ACh-induced current, and the ACh-induced current response could be inhibited by pre-incubation with pertussis toxin (PTX). It is concluded that the m4 muscarinic receptor is coupled to an inwardly rectifying potassium conductance via a PTX sensitive G-protein.

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N-(2-methoxyphenyl) benzenesulfonamide, a novel regulator of neuronal G protein-gated inward rectifier K<sup>+</sup> channels
2017, European Journal of Pharmacology
Citation Excerpt :
The results shown in Figs. 1 and 2 indicate that MPBS regulates the GIRK channel activated by somatostatin. In addition to SSTRs, AtT20 cells endogenously express muscarinic receptors that couple to GIRK channels through Gi/Go proteins (Dousmanis and Pennefather, 1992; Jones, 1992). Previous studies have also demonstrated that cannabinoids can activate GIRK channels in AtT20 cells permanently transfected with the CB1 receptor (Mackie et al., 1995).
G protein-gated inward rectifier K⁺ (GIRK) channels are members of the super-family of proteins known as inward rectifier K⁺ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K⁺ channel, another member of the Kir channel family, is regulated by sulfonamide drugs. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the somatostatin type-2 and -5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments, application of MPBS both shifted the somatostatin concentration-response curve (EC₅₀ = 3.5 nM [control] vs.1.0 nM [MPBS]) for GIRK channel activation and increased the maximum GIRK current measured with 100 nM somatostatin. However, GIRK channel activation was not observed when MPBS was applied to the cells in the absence of somatostatin. While the MPBS structural analog 4-fluoro-N-(2-methoxyphenyl) benzenesulfonamide also augmented the somatostatin-induced GIRK fluorescent signal, no increase in the signal was observed with the sulfonamides tolbutamide, sulfapyridine and celecoxib. In conclusion, MPBS represents a novel prototypic GPCR-dependent regulator of neuronal GIRK channels.
Blockade of different muscarinic receptor subtypes changes the equilibrium between excitation and inhibition in rat visual cortex
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We have shown that cortical acetylcholine modulates the balance between excitation and inhibition evoked in layer 5 pyramidal neurons of rat visual cortex [Lucas-Meunier E, Monier C, Amar M, Baux G, Frégnac Y, Fossier P (2009) Cereb Cortex 19:2411–2427]. Our aim is now to establish a functional basis for the role of the different types of muscarinic receptors (MRs) on glutamate fibers and on GABAergic interneurons and to analyse their contribution to the modulation of excitation-inhibition balance in the rat visual cortex. To ascertain that there was a basis for our functional study, we first checked for the presence of the various MR subtypes by single cell RT-PCR and immunolabeling experiments. Then, recording the composite responses in layer 5 pyramidal neurons to layer 1–2 stimulation (which also recruits cholinergic fibers) in the presence of specific antagonists of the different types of MR allowed us to determine their modulatory role. We show that the specific blockade of the widely distributed M1R (with the mamba toxin, MT7) induced a significant increase in the excitatory conductance without modifying the inhibitory conductance, pointing to a localization of M1R on glutamatergic neurons where their activation would decrease the release of glutamate. From our functional results, M2/M4Rs appear to be located on glutamatergic neurons afferent to the recorded layer 5 pyramidal neuron and they decrease glutamate release. The extended distribution of M4Rs in the cortex compared to the restricted distribution of M2R (layers 3–5) is in favour of a major role as a modulator of M4R. The selective antagonist of M3Rs, 4-DAMP, decreased the inhibitory conductance, showing that activated M3Rs increase the release of GABA and thus are located on GABAergic interneurons. The activation of the different types of MRs located either on glutamatergic neurons or on GABAergic interneurons converges to reinforce the dominance of inhibitory inputs thus decreasing the excitability of layer 5 pyramidal neurons.
Corticotropin releasing factor-induced ERK phosphorylation in AtT20 cells occurs via a cAMP-dependent mechanism requiring EPAC2
2010, Neuropharmacology
Citation Excerpt :
To show efficacy of PTX and BAPTA-AM, their effect on carbachol-induced ERK phosphorylation was investigated (Fig. 3C). Carbachol has been shown to trigger Gi protein signalling (Jones, 1992) a mechanism that could enhance ERK phosphorylation (Lakatos et al., 2005). Indeed, stimulation of AtT20 cells with carbachol augmented the phospho-ERK signal and the effect was abolished in the presence of both PTX and BAPTA-AM (P < 0.05; Fig. 3C).
CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression of pro-opiomelanocortin, a key precursor molecule in the hypothalamic pituitary adrenal axis. In AtT20 cells, CRF signalling has been investigated but the mechanism behind CRF-induced ERK activity is not fully understood. This paper elucidates the signalling cascade involved in this phenomenon.
Involvement of CRF₁ receptor on ERK phosphorylation was shown by using CRF and urocortin 1. The lack of inhibitory effect of pertussis toxin and BAPTA-AM excluded involvement of G_i-coupling and calcium mobilization respectively. In contrast, the process is suggested to be driven by cAMP since treatment of AtT20 cells with forskolin triggered strong ERK phosphorylation. Treatment with PKA inhibitors had a minor effect on CRF-induced ERK signalling while phosphorylation of CREB was completely abolished. This ruled out involvement of PKA and suggested a role for exchange protein directly activated by cAMP (EPAC). Moreover, an activator of EPACs 8-(4-methoxyphenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate mimicked CRF-induced ERK phosphorylation. Gene expression analysis showed high levels of EPAC2 mRNA and protein but low levels of EPAC1. Knockdown of EPAC2 expression by the use of specific siRNAs abolished CRF- and forskolin-induced ERK phosphorylation. The current study demonstrates a clear cAMP-dependent but PKA-independent mechanism underlying CRF-induced ERK activity that proceeds via EPAC2 signalling. Further research will provide more insight in the role of EPAC2 in CRF signalling.
GoLoco motif peptides as probes of Gα subunit specificity in coupling of G-protein-coupled receptors to ion channels
2004, Methods in Enzymology
Biochemical and structural studies of signaling proteins have revealed critical features of peptide motifs at the interaction surfaces between proteins. Such information can be used to design small peptides that can be used as functional probes of specific interactions in signaling cascades. This article describes the use of a novel domain (the GoLoco motif) found in several members of the regulators of G-protein signaling (RGS) protein family to probe the specificity of Gα subunit involvement in the coupling of dopamine and somatostatin receptors to ion channels in the AtT20 neuroendocrine cell line. Peptides encoding the GoLoco motifs of RGS12 and AGS3 were perfused into single cells during electrical recording of agonist-induced current responses by whole cell patch clamp methods. The particular sequences chosen have been demonstrated to bind selectively to the GDP-bound form of Gα_i, but not Gα_o, and preclude association of Gβγ and Gα_i subunits. A functional manifestation of this property is observed in the progressive uncoupling of D2 dopamine receptors and Kir3.1/3.2 channels with repeated agonist application. Similar uncoupling is not observed with somatostatin receptors nor with D2 receptors coupling to calcium channels, suggesting Gα subunit specificity in these signaling pathways. Motifs found in other proteins in the GPCR signaling machinery may also prove useful in assessing G-protein signaling specificity and complexity in single cells in the future.
Hyperpolarization-activated inward potassium and calcium-sensitive chloride currents in beating pacemaker insect neurosecretory cells (dorsal unpaired median neurons)
1999, Neuroscience
Hyperpolarization-activated inward currents were studied in single adult cockroach Periplaneta americana pacemaker neurosecretory cells, identified as dorsal unpaired median neurons using the whole-cell patch-clamp technique. Under current clamp, injection of negative current produced a hyperpolarization of the cell membrane with a sag in the membrane potential toward the resting value. Under voltage clamp, the whole-cell current–voltage relationship exhibited an unexpected biphasic aspect. The global hyperpolarization-activated inward current could be dissociated by means of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid and tetraethylammonium chloride sensitivity, ionic selectivity, voltage dependence and activation threshold as inward potassium and calcium-sensitive chloride currents. The inward potassium current was activated around −80 mV. The reversal potential followed the potassium equilibrium potential when the extracellular potassium concentration was raised. This current was not dependent on the external sodium concentration and was sensitive to 10 mM tetraethylammonium chloride or 5 mM barium chloride. The hyperpolarization-activated inward calcium-sensitive chloride current was activated in a range of potential 20 mV more positive than the potassium current. The estimated reversal potential (−71 mV) was very close to the equilibrium potential for chloride ions (−73 mV). Intracellularly applied 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and external application of 1 mM zinc chloride, calcium-free saline or high concentrations of intracellular 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetate blocked the inward chloride current. Current-clamp experiments indicated that the inward potassium current accounted for inward rectification of dorsal unpaired median neurons.
Our findings report, for the first time in pacemaker neurosecretory cells, the co-existence of two distinct hyperpolarization-activated inward currents which have specialized function in pacemaker activity.
Muscarinic M<inf>1</inf> and M<inf>3</inf> receptors are present and increase intracellular calcium in adult rat anterior pituitary gland
1999, Brain Research Bulletin
Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]diphenyl-acetoxy-N-methyl-piperidine ([³H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [³H]QNB and [³H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M₁, M₃ subtypes selective antagonist) > atropine (general) > pirenzepine (M₁). Methoctramine (M₂) didn’t antagonise the [³H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca²⁺ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M₁ and M₃ muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.

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M4 muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT20 cells

Abstract

Localization of mRNAs encoding the α-subunits of signal-transducing G-proteins within rat brain and among peripheral tissues

FEBS Lett.

Localization of a family of muscarinic acetylcholine receptor genes

J. Neurosci.

Single-step method of RNA isolation by acid guanidinium thiocyanate

Anal. Biochem.

Muscarinic modulation of cardiac rate at low acetylcholine concentrations

Science

Immunological detection of muscarinic receptor subtype proteins (m1–m5) in rabbit peripheral tissues

Mol. Pharmacol.

Characterization of the inwardly-rectifying conductances in AtT-20 cells

Biophys. J., Abstr.

Improved patchclamp techniques for high resolution current recording from cells and cell-free membrane patches

Pflügers Arch.

Muscarinic acetylcholine receptors