Different sensitivities to agonist of muscarinic acetylcholine receptor subtypes

Muscarinic acetylcholine receptor (mAChR) III expressed in Xenopus oocytes, like mAChR I, mediates activation of a Ca²⁺-dependent Cl⁻ current, whereas mAChR IV, like mAChR II, principally induces activation of Na⁺ and K⁺ currents in a Ca²⁺-independent manner. mAChR III has a sensitivity to agonist of about one order of magnitude higher than that of mAChR I in mediating the Ca²⁺-dependent current response in Xenopus oocytes and in stimulating phosphoinositide hydrolysis in NG108-15 neuroblastoma-glioma hybrid cells. The agonist-binding affinity of mAChR III is also about one order of magnitude higher than that of mAChR I.     

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.     

Ca entry through the store-mediated pathway directly activates only the K current but the subsequent Ca release from the store activates both K and Cl currents in submandibular gland acinar cells of the rat

The store-mediated Ca²⁺ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca²⁺ activated ionic membrane currents. In the cells where intracellular Ca²⁺ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca²⁺-free extracellular condition, an employment of external Ca²⁺ in the absence of ACh caused a sustained increase of the K⁺ current without affecting the Cl⁻ current. A renewed ACh challenge without external Ca²⁺ caused repetitive spikes of both K⁺ and Cl⁻ currents due to the Ca²⁺ release. SK & F 96365 inhibited the generation of the sustained K⁺ current and refilling of the Ca²⁺ store following the Ca²⁺ readmission. It is suggested that the Ca²⁺ enters the cell through the store-mediated pathway near the K⁺ channels and is taken up by the store. Thus, only Ca²⁺ released from the store can activate both the K⁺ and Cl⁻ currents.     

Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes

The tissue distribution of the mRNAs encoding muscarinic acetylcholine receptors (mAChRs) I, II, III and IV has been investigated by blot hybridization analysis with specific probes. This study indicates that exocrine glands contain both mAChR I and III mRNAs, whereas smooth muscles contain both mAChR II and III mRNAs. All four mAChR mRNAs are present in cerebrum, whereas only mAChR II mRNA is found in heart.     

Inhibition of post-synaptic Kv7/KCNQ/M channels facilitates long-term potentiation in the hippocampus

Activation of muscarinic acetylcholine receptors (mAChR) facilitates the induction of synaptic plasticity and enhances cognitive function. In the hippocampus, M(1) mAChR on CA1 pyramidal cells inhibit both small conductance Ca(2+)-activated KCa2 potassium channels and voltage-activated Kv7 potassium channels. Inhibition of KCa2 channels facilitates long-term potentiation (LTP) by enhancing Ca(2+)calcium influx through postsynaptic NMDA receptors (NMDAR). Inhibition of Kv7 channels is also reported to facilitate LTP but the mechanism of action is unclear. Here, we show that inhibition of Kv7 channels with XE-991 facilitated LTP induced by theta burst pairing at Schaffer collateral commissural synapses in rat hippocampal slices. Similarly, negating Kv7 channel conductance using dynamic clamp methodologies also facilitated LTP. Negation of Kv7 channels by XE-991 or dynamic clamp did not enhance synaptic NMDAR activation in response to theta burst synaptic stimulation. Instead, Kv7 channel inhibition increased the amplitude and duration of the after-depolarisation following a burst of action potentials. Furthermore, the effects of XE-991 were reversed by re-introducing a Kv7-like conductance with dynamic clamp. These data reveal that Kv7 channel inhibition promotes NMDAR opening during LTP induction by enhancing depolarisation during and after bursts of postsynaptic action potentials. Thus, during the induction of LTP M(1) mAChRs enhance NMDAR opening by two distinct mechanisms namely inhibition of KCa2 and Kv7 channels.     

Alteration of channel characteristics by exchange of pore-forming regions between two structurally related Ca2+ Channels

Several types of structurally homologous high voltage-gated Ca²⁺ channels (L-, P-and N-type) have been identified via biochemical, pharmacological and electrophysiological techniques. Among these channels, the cardiac L-type and the brain BI-2 Ca²⁺ channel display significantly different biophysical properties. The BI-2 channel exhibits more rapid voltage-dependent current activation and inactivation and smaller single-channel conductance compared to the L-type Ca²⁺ channel. To examine the molecular basis for the functional differences between the two structurally related Ca²⁺ channels, we measured macroscopic and single-channel currents from oocytes injected with wild-type and various chimeric channel ₁ subunit cRNAs. The results show that a chimeric channel in which the segment between S5-SS2 in repeat IV of the cardiac L-type Ca²⁺ channel, was replaced by the corresponding region of the BI-2 channel, exhibited macroscopic current activation and inactivation time-courses and single-channel conductance, characteristic of the BI-2 Ca²⁺ channel. The voltage-dependence of steady-state inactivation was not affected by the replacement. Chimeras, in which the SS2-S6 segment in repeat III or IV of the cardiac channel was replaced by the corresponding BI-2 sequence, exhibited altered macroscopic current kinetics without changes in single-channel conductance. These results suggest that part of the S5-SS2 segment plays a critical role in determining voltage-dependent current activation and inactivation and single-channel conductance and that the SS2-S6 segment may control voltage-dependent kinetics of the Ca²⁺ channel.     

Spatiotemporal calcium signaling in a<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> cell line stably expressing a<Emphasis Type="Italic"> Drosophila</Emphasis> muscarinic acetylcholine receptor

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca²⁺]_i) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca²⁺]_i transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.     

Muscarinic Receptor Stimulation Activates a Ca<Superscript>2+</Superscript>-dependent Cl<Superscript>-</Superscript> Conductance in Rat Distal Colon

Recently, it was observed that the acetylcholine analogue carbachol induces a transient stimulation of an apical Cl(-) conductance in basolaterally depolarized rat distal colonic epithelium (Schultheiss et al., 2003). The further characterization of this conductance was the aim of the present study. All experiments were performed at basolaterally depolarized tissues (111.5 mmol.l(-1) KCl buffer at the serosal side); in the absence of a K(+) gradient, a Cl(-) current was driven across the apical membrane (107 mmol.l(-1) K gluconate/4.5 mmol.l(-1) KCl buffer on the mucosal side). Under these conditions, carbachol evoked an atropine-sensitive biphasic change in short-circuit current (I(SC)), consisting of a transient increase followed by a long-lasting decrease, suggesting a stimulation of apical Cl(-) conductance followed by an inhibition. This conductance was inhibited by SITS, but was resistant against glibenclamide, a blocker of CFTR. The carbachol-induced I(SC) was dependent on the presence of mucosal Ca(2+). Ionomycin, a Ca(2+) ionophore, mimicked the effect of carbachol. An antibody against bovine Ca(2+)-activated Cl(-) channel ClCa 1 stained rat colonic epithelial cells both at the cell membrane as well as intracellularly, suggesting that the action of Ca(2+) may be caused by a stimulation of a ClC a-type anion channel. The activation of apical Cl(-) conductance by carbachol was resistant against any blockers of the phospholipase C/IP3/protein kinase C pathway tested (e.g., U-73122, 2-ABP, Li(+), staurosporine), but was inhibited by the NO-synthase blocker L: -NNA. Vice versa, NO-donating compounds such as GEA 3162 or sodium nitroprusside evoked a transient increase of I(SC). Consequently, NO seems to be involved in the transient stimulation of apical Ca(2+)-dependent Cl(-) conductance after muscarinic receptor stimulation.     

Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.     

Graded response to GABA by native extrasynaptic GABA receptors

GABA is the main inhibitory neurotransmitter in the mammalian CNS. GABA in the brain is commonly associated with a fast, point-to-point form of signalling called synaptic transmission (phasic inhibition), but there is growing evidence that GABA participates in another, slower and more diffuse form of signalling often referred to as tonic inhibition. Unresolved questions regarding tonic neuronal inhibition concern activation and functional properties of extrasynaptic GABAA receptors (GABARex) present on neurones. Extrasynaptic receptors are exposed to submicromolar GABA concentrations and may modulate the overall excitability of neurones and neuronal networks. Here, we examined GABA-activated single-channel currents in dentate gyrus granule neurones in rat hippocampal slices. We activated three types (I, II, III) of GABARex channels by nanomolar GABA concentrations (EC50 I: 27 +/- 12; II: 4 +/- 3; III: 43 +/- 19 nm). The channels opened after a delay and the single-channel conductance was graded (gammamax I: 61 +/- 3; II: 85 +/- 8, III: 40 +/- 3 pS). The channels were differentially modulated by 1 microm diazepam, 200 nm zolpidem, 1 microm flumazenil and 50 nm THDOC (3alpha, 21-dihydroxy-5alpha-pregnan-20-one), consistent with the following minimal subunit composition of GABARex I alpha1betagamma2, GABARex II alpha4betagamma2 and GABARex III alphabetadelta channels.     

Stimulation of D-2 Dopamine Receptors Decreases the Evoked In Vitro Release of [3H] Acetylcholine from Rat Neostriatum: Role of K+ and Ca2+

Reportedly, stimulation of D-2 dopamine receptors inhibits the depolarization-induced release of acetylcholine from the neostriatum in a cyclic AMP-independent manner. In the present study, we investigated the role of K+ and Ca2+ in the D-2 receptor-mediated inhibition of evoked [3H]acetylcholine release from rat striatal tissue slices. It is shown that the D-2 receptor-mediated decrease of K+-evoked [3H]acetylcholine release is not influenced by the extracellular Ca2+ concentration. However, increasing extracellular K+, in the presence and absence of Ca2+, markedly attenuates the effect of D-2 stimulation on the K+-evoked [3H]acetylcholine release. Furthermore, it is shown that activation of D-2 receptors in the absence of Ca2+ also inhibits the veratrine-evoked release of [3H]acetylcholine from rat striatum. These results suggest that the D-2 dopamine receptor mediates the decrease of depolarization-induced [3H]acetylcholine release from rat striatum primarily by stimulation of K+ efflux (opening of K+ channels) and inhibition of intracellular Ca2+ mobilization.     

Upregulation of mRNA Encoding the M5 Muscarinic Acetylcholine Receptor in Human T- and B-Lymphocytes During Immunological Responses

Lymphocytes possess an independent, non-neuronal cholinergic system. Moreover, both T- and B-lymphocytes express multiple muscarinic acetylcholine receptors (mAChR). To obtain a better understanding of the regulatory mechanisms governing mAChR gene expression in the lymphocytic cholinergic system, we examined the effects of lymphocyte activation on expression of mAChR mRNA. Stimulation of T- and B-lymphocytes, respectively, with T-cell activator phytohemagglutinin and B-cell activator Staphylococcus aureus Cowan I upregulated M₅ mAChR mRNA expression in the CEM human leukemic T-cell line and in the Daudi B-cell line, which served as models of lymphocytes. In striking contrast, M₃ and M₄ mAChR mRNA expression was not affected in either cell line. Nonetheless, stimulating lymphocytes with phorbol 12-myristate 13-acetate, a protein kinase C activator, plus ionomycin, a calcium ionophore, upregulated expression of both M₃ and M₅ mAChR mRNA. This represents the first demonstration that immunological stimulation leads to M₅ mAChR gene expression in lymphocytes.     

Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel.

In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.     

Down-regulation of brain muscarinic cholinergic receptor promoted by diacylglycerols and phorbol ester

Sustained agonist stimulation induces an asymmetric down-regulation of brain muscarinic acetylcholine receptor (mAChR): 43±2% in the right and 26±2% in the left cerebral hemisphere, respectively (Ref. 1). In order to determine the possible involvement of endogenous diacylglycerols produced under muscarinic stimulation in the down-regulation phenomenon, here we have studied the effects of synthetic diacylglycerols and a phorbol ester on cells dissociated from rat cerebral cortex. Oleoylacetylglycerol decreased the amount of cell-surface mAChR by 37±2% and 25±2% in right and left cerebral cortex, respectively. Long-term treatment with phorbol dibutyrate also produced internalization of the mAChR (25±1.5% and 33±2% in right and left cortical cells, respectively). These changes occurred without modification of the Kd_app for the selective antagonist pirenzepine. The action of calcium ions was also studied using incubation of cells with the ionophore A23187. No changes were observed in the amount of mAChR detected at the plasma membrane with the ionophore alone, but when used in combination with phorbol dibutyrate and the agonist carbamylcholine a sinergistic decrease in mAChR was apparent. It is concluded that long-term exposure to exogenously added diacyglycerols and phorbol ester significantly reduces the amount of mAChR detected at the plasma membrane and abolishes the asymmetry of the down-regulation phenomenon observed under specific muscarinic stimulation, suggesting that diacylglycerols may be one of the factors responsible for such asymmetry.Abbreviations used A23187 ionophore A23187 - ATRO atropine - CARB carbamoylcholine - DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer - PZ pirenzepine - LCC left cerebral cortex - mAChR muscarinic acetylcholine receptor - OAG oleoylacetylglycerol - PDB phorbol dibutyrate - RCC right cerebral cortex     

PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule neuron death.     

Single-channel and whole-cell currents evoked by acetylcholine in dissociated sympathetic neurons of the rat

Single acetylcholine-activated channels have been recorded from neurons dissociated from the sympathetic chain of 17-21 day old rats. The mean single channel conductance is 35 pS in normal medium containing 1 mM calcium, and 51 pS in the absence of calcium. The measured current amplitudes are about five times more variable than at the frog endplate, at least in part because the current, while the channel is open, is much noisier than when it is shut. Single activations of the receptor by acetylcholine (ACh) produce a burst of openings; the distribution of the burst length has two components, the longer of which is of primary importance in synaptic transmission. Whole-cell currents, in response to ACh (up to 30 microM), show strong inward rectification with no outward current being detectable. This phenomenon is similar whether the intracellular ion is sodium or cesium, whether or not divalent cations are present, and whether or not atropine is present. Nevertheless, outward single-channel currents (of normal conductance) are detectable in isolated outside-out patches.     

Calcium-dependent Enhancement of Depletion-activated Calcium Current in Jurkat T Lymphocytes

We have obtained evidence that the Ca²⁺-selective current activated by Ca²⁺ store depletion (Ca²⁺ release-activated Ca²⁺ current; I _crac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca²⁺ itself. Whole cell patch clamp experiments employed high cytosolic Ca²⁺-buffering conditions to passively deplete Ca²⁺ stores. Rapidly switching to nominally Ca²⁺-free extracellular buffer instantaneously reduced I _crac measured at −100 mV to leak current level. Unexpectedly, readmission of 2 mm Ca²⁺ instantaneously restored only 38 ± 5% (mean ±sem; n = 9) of the full I _crac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca²⁺ chelators did not alter this process, and inorganic I _crac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca²⁺: introduction of the permeant ions, Ba²⁺ or Sr²⁺, failed to invoke time-dependent I _crac reappearance. Moreover, equimolar substitution of Ba²⁺ for Ca²⁺ initially produced Ba²⁺ current of similar magnitude to the full Ca²⁺ current, but the Ba²⁺ current decayed monotonically to <50% of its initial amplitude in <20 sec. Conversely, return to Ca²⁺ produced a time-dependent increase in I _crac to its larger Ca²⁺ permeation level. Thus Ca²⁺ appears to selectively promote a reversible transition of I _crac that results in larger current flux, and at least partially explains the selectivity of this current for Ca²⁺ over other divalent ions. Received: 30 August 1995/Revised: 7 November 1995