Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.     

Muscarinic toxin 7 selectivity is dictated by extracellular receptor loops

Muscarinic toxin 7 (MT7) is a mamba venom protein antagonist with extremely high selectivity for the M1 muscarinic acetylcholine receptor. To map the sites for the interaction of MT7 with muscarinic receptors we have used chimeric M1:M3 receptors and site-directed mutagenesis of the M3 and M4 receptor subtypes. Two Glu residues in M1, one in extracellular loop 2 and one in extracellular loop 3, were found to be important for the high affinity binding of MT7. Substitution of the corresponding Lys residues in the M3 receptor with Glu converted the M3 mutant to an MT7 binding receptor, albeit with lower affinity compared with M1. A Phe --> Tyr substitution in extracellular loop 2 of M3 together with the 2 Glu mutations generated a receptor with an increased MT7 affinity (apparent Ki = 0.26 nM in a functional assay) compared with the M1 receptor (apparent Ki = 1.31 nM). The importance of the identified amino acid residues was confirmed with a mutated M4 receptor constructs. The results indicate that the high selectivity of MT7 for the M1 receptor depends on very few residues, thus providing good prospects for future design and synthesis of muscarinic receptor-selective ligands.     

Transient hypoxia induces sequestration of M1 and M2 muscarinic acetylcholine receptors

Oxidative stress has been implicated in impairing muscarinic acetylcholine receptor (mAChR) signaling activity. It remains unclear, however, whether alterations in the cell surface distribution of mAChRs following oxidative stress contribute to the diminished mAChR signaling activity. We report here that M1 and M2 mAChRs, stably expressed in Chinese hamster ovary cells, undergo sequestration following transient hypoxic-induced oxidative stress (2% O2). Sequestration of M1 and M2 mAChRs following transient hypoxia was associated with an increase in phosphorylation of these receptors. Over-expression of a catalytically inactive G protein-coupled receptor kinase 2 (GRK2 K220R) blocked the increased phosphorylation and sequestration of the M2, but not M1, mAChRs following transient hypoxia. Hypoxia induced phosphorylation and sequestration of the M1 mAChR was, however, blocked by over-expression of a catalytically inactive casein kinase 1 alpha (CK1alpha K46R). These results are the first demonstration that M1 and M2 mAChRs undergo sequestration following transient hypoxia. The data suggest that increased phosphorylation of M1 and M2 mAChRs underlies the mechanism responsible for sequestration of these receptors following transient hypoxia. We report here that distinct pathways involving CK1alpha and GRK2 mediated sequestration of M1 and M2 mAChRs following transient hypoxic-induced oxidative stress.     

AFDX-116 discriminates between muscarinic M2 receptors of the heart and the iris smooth muscle

Summary Studies with the atypical muscarinic antagonist pirenzepine provide convincing evidence for the classification of muscarinic acetylcholine receptors (mAChRs) into two subtypes, M₁ and M₂. The present study examines the heterogeneity of the M₂ subtype employing the newly developed competitive muscarinic antagonist, AFDX-116. Comparison of the binding affinities of pirenzepine, atropine, and AFDX-116 to mAChRs in microsomes from the rabbit cerebral cortex, heart, and iris smooth muscle shows that iris mAChRs, which are pharmacologically of the M₂ subtype, can be distinguished from M₂ cardiac receptors based on their affinity for AFDX-116. These results are consistent with the hypothesis that the M₂ receptor subtype consists of a heterogeneous population of receptors.Abbreviations mAChRs Muscarinic Acetylcholine Receptors - CCh Carbachol - NMS N-Methylscopolamine - AFDX-116 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepine-6-one     

Molecular events associated with the regulation of signaling by M2 muscarinic receptors

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.     

CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C‐terminal regions of second intracellular loops. This binding relies on Ca²⁺ activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca²⁺ recruits CaMKIIα to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity‐dependent manner. Our data identify a new model of protein–protein interactions. In a Ca²⁺‐sensitive manner, CaMKIIα regulates M4R efficacy and controls the acetylcholine–dopamine balance in the basal ganglia and also the dynamics of movement.     

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

Background information. The idea that GPCRs (G‐protein‐coupled receptors) may exist as homo‐ or hetero‐oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM₁ (human M₁ muscarinic acetylcholine receptor subtype). Results. We analysed the hM₁ oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native‐PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO‐M1 cells (Chinese‐hamster ovary cells expressing muscarinic M₁ receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M₁ receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M₁ receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Conclusions. Our results suggest that MT7 binds to a dimeric form of hM₁ receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre‐existing muscarinic receptor homodimers.     

Identification of a basolateral sorting signal for the M3 muscarinic acetylcholine receptor in Madin-Darby canine kidney cells

Muscarinic acetylcholine receptors (mAChRs) can be differentially localized in polarized cells. To identify potential sorting signals that mediate mAChR targeting, we examined the sorting of mAChRs in Madin-Darby canine kidney cells, a widely used model system. Expression of FLAG-tagged mAChRs in polarized Madin-Darby canine kidney cells demonstrated that the M(2) subtype is sorted apically, whereas M(3) is targeted basolaterally. Expression of M(2)/M(3) receptor chimeras revealed that a 21-residue sequence, Ser(271)-Ser(291), from the M(3) third intracellular loop contains a basolateral sorting signal. Substitution of sequences containing the M(3) sorting signal into the homologous regions of M(2) was sufficient to confer basolateral localization to this apical receptor. Sequences containing the M(3) sorting signal also conferred basolateral targeting to M(2) when added to either the third intracellular loop or the C-terminal cytoplasmic tail. Furthermore, addition of a sequence containing the M(3) basolateral sorting signal to the cytoplasmic tail of the interleukin-2 receptor alpha-chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M(3) basolateral sorting signal is dominant over apical signals in M(2) and acts in a position-independent manner. The M(3) sorting signal represents a novel basolateral targeting motif for G protein-coupled receptors.     

Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.     

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M(2) mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.     

The Cysteine Residue in the Carboxyl-Terminal Domain of the m2 Muscarinic Acetylcholine Receptor Is Not Required for Receptor-Mediated Inhibition of Adenylate Cyclase

Muscarinic acetylcholine receptors (mAChRs) share with many other receptors of the guanine nucleotide-binding protein-coupled receptor family a highly conserved cysteine residue in the putative cytoplasmic carboxyl-terminal region of the protein. Because elimination of this cysteine in the beta 2-adrenergic receptor has been reported to decrease functional responsiveness, we determined if this cysteine residue is essential for mAChR-effector coupling by replacing Cys457 of the m2 mAChR with glycine and expressing wild-type and mutant receptor in Chinese hamster ovary (CHO) cells. The mutant and wild-type receptors exhibited similar affinities for binding of muscarinic ligands. In addition, the mutation did not affect cell surface localization or receptor-mediated inhibition of adenylate cyclase. These results indicate that the cysteine residue in the carboxyl-terminal domain of the m2 mAChR is not required for ligand binding or mAChR-mediated inhibition of adenylate cyclase in CHO cells.     

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional “three-finger” snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by “three-finger” snake neurotoxins.     

Charged amino acids required for signal transduction by the m3 muscarinic acetylcholine receptor.

The five muscarinic acetylcholine receptor (mAChR) subtypes, termed m1-m5, transduce agonist signals across the plasma membrane by activating guanine nucleotide binding (G) proteins. The large cytoplasmic domain joining the fifth and sixth transmembrane segments of mAChRs plays a critical role in controlling the specificity of G protein coupling. In this study, we determined which sequences within this domain are required for activation of signaling by the m3 mAChR. By measuring the ability of normal and mutant m3 mAChRs to couple to the G protein pathway leading to activation of phospholipase C and Ca(2+)-dependent chloride currents in RNA-injected Xenopus oocytes, we found that two clusters of charged residues near the fifth and sixth transmembrane segments were required for normal signaling; furthermore, the position of these sequences was critical for their function. Finally, analysis of deletion mutant m3 mAChRs confirmed the importance of these sequences; receptors containing as few as 22 out of 239 amino acids of the cytoplasmic domain were fully active in signaling if they included the critical charged residues. Sequence comparisons suggest that similar charged sequences may be required for signal transduction by many G protein-coupled receptors.     

Activation of muscarinic M4 receptor augments NGF-induced pro-survival Akt signaling in PC12 cells

Survival or death of neurons during development is mediated by the integration of a diverse array of signal transduction cascades that are controlled by the availability and acquisition of neurotrophic factors and agonists acting at G protein-coupled receptors (GPCRs). Recent studies have demonstrated that GPCRs can modulate signals elicited by receptor tyrosine kinases (RTK) and vice versa. Here, we examined the activity of pro-survival Akt kinase, in response to stimulation by muscarinic acetylcholine receptors (mAChRs) and co-activation with the nerve growth factor (NGF) receptor in PC12 cells endogenously expressing Gi-coupled M4 mAChR and Gq-coupled M1 and M5 mAChRs. Western blotting analysis using a phosphospecific anti-Akt antibody revealed a dose- and time-dependent increase in Akt phosphorylation in cells stimulated with mAChR specific agonist carbachol (CCh). Co-stimulation with CCh and NGF resulted in augmentation of Akt activity in a pertussis toxin (PTX)-sensitive manner, suggesting that M4 mAChR, but not M1 and M5 mAChRs, was associated with this synergistic Akt activation. The use of transducin as a Gbetagamma scavenger indicated that Gbetagamma subunits rather than Galphai/o acted as the signal transducer. Additional experiments showed that CCh treatment augmented NGF-induced phosphorylation and degradation of the Akt-regulated translation regulator tuberin. This augmentation was also inhibited by PTX pre-treatment or overexpression of transducin. Finally, co-stimulation of PC12 cells with CCh and NGF resulted in enhancement of cell survival. This is the first study that demonstrates the augmentation effect between M4 mAChR and NGF receptor, and the regulatory role of mAChR on tuberin.     

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light-adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light-adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype-specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)-induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol-induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.     

昆虫毒蕈碱型乙酰胆碱受体的研究进展

毒蕈碱型乙酰胆碱受体(Muscarinic Acetylcholine Receptors,mAChRs)是昆虫神经系统中一类重要的G蛋白偶联受体.昆虫mAChRs可以分为A、B、C型三大类,它们通过偶联不同的G蛋白激活不同的第二信使,完成信号转导过程,从而发挥其功能.mAChRs参与调控昆虫多种生理反应和行为过程,如...     

Regulation of muscarinic acetylcholine receptor sequestration and function by beta-arrestin

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta2-adrenergic receptor have demonstrated that beta-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of beta-arrestin in mAChR sequestration, we determined the effect of overexpressing beta-arrestin-1 and the dominant-negative inhibitor of beta-arrestin-mediated receptor sequestration, beta-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a beta-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.     

Release-enhancing pre-synaptic muscarinic and nicotinic receptors co-exist and interact on dopaminergic nerve endings of rat nucleus accumbens

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR–mAChR agonists acetylcholine (ACh) and carbachol provoked [³H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 μmol/L, together with 3, 10, or 100 μmol/L (−)nicotine provoked synergistic effect on [³H]DA overflow. The [³H]DA overflow elicited by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was reduced by atropine down to the release produced by (−)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (−)nicotine/oxotremorine evoked [³H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (−)nicotine/oxotremorine. Similarly to (−)nicotine, the selective α4β2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, α4β2 nAChRs exert a permissive role on the releasing function of reportedly M₅ mAChRs co-existing on the same dopaminergic nerve endings.     

Functional effects of protein kinase C-mediated phosphorylation of chick heart muscarinic cholinergic receptors.

Muscarinic cholinergic receptors (mAChR) purified from chick heart were phosphorylated by protein kinase C (PKC) and reconstituted with the purified GTP-binding regulatory protein Go. The effects of PKC phosphorylation on the interaction of mAChR with Go were assessed by monitoring for agonist-stimulated guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding to Go, agonist-stimulated GTPase activity of Go, and the capability of Go to induce high affinity agonist binding to mAChR. Both the receptor-stimulated GTP gamma S binding and GTPase activity of Go were markedly diminished as a result of PKC-mediated phosphorylation of the mAChR, whereas the ability of Go to induce high affinity agonist binding to the receptors was unaffected. When mAChR were first reconstituted with Go and then subjected to phosphorylation with PKC, a complete inhibition of the phosphorylation of mAChR by PKC was observed. The inhibitory effect of Go on mAChR phosphorylation was concentration-dependent and was prevented by the presence of GTP gamma S in the reaction mixtures. Taken together, these results indicate that the phosphorylation of mAChR by PKC modulates receptor/G-protein interactions and that the ability of the receptors to act as substrates for PKC may be regulated by receptor/G-protein interactions.