Microinjected Xenopus Oocytes Research Articles

The SARS-CoV-2 ORF6 protein inhibits nuclear export of mRNA and spliceosomal U snRNA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19). SARS-CoV-2 infection suppresses host innate immunity and impairs cell viability. Among the viral proteins, ORF6 exhibits potent interferon (IFN) antagonistic activity and cellular toxicity. It also interacts with the RNA export factor RAE1, which bridges the nuclear pore complex and nuclear export receptors, suggesting an effect on RNA export. Using the Xenopus oocyte microinjection system, I found that ORF6 blocked the export of not only mRNA but also spliceosomal U snRNA. I further demonstrated that ORF6 affects the interaction between RAE1 and nuclear export receptors and inhibits the RNA binding of RAE1. These effects of ORF6 may cumulatively block the export of several classes of RNA. I also found that ORF6 binds RNA and forms oligomers. These findings provide insights into the suppression of innate immune responses and the reduction in cell viability caused by SARS-CoV-2 infection, contributing to the development of antiviral drugs targeting ORF6.

A Brief History of Xenopus in Biology.

Xenopus is one of the premier model systems to study cell and developmental biology in vivo in vertebrates. Here we briefly review how this South African frog came to be favored by a large community of scientists after the explosive growth of molecular biology and examine some of the original discoveries arising from this sturdy frog. Experimental embryology started in Rana but developed in newt embryos for historical reasons. A long lineage of mentorship, starting with Theodor Boveri, Hans Spemann, Fritz Baltzer, Ernst Hadorn, and Michail Fischberg, used newt embryos. In Oxford, Fischberg made the transition to Xenopus laevis because it was widely available for human pregnancy tests and laid eggs year-round, and he fortuitously isolated a one-nucleolus mutant. This mutant allowed nuclear transfer experiments showing that genetic information is not lost during cell differentiation and the demonstration that the nucleolus is the locus of transcription of the large ribosomal RNAs. With the advent of DNA cloning, the great equalizer among all fields of biology, microinjected Xenopus oocytes became an indispensable tool, providing the first living-cell mRNA translation, polymerase II and III transcription, and coupled transcription-translation systems in eukaryotes. Xenopus embryos provide abundant material to study the earliest signaling events during vertebrate development and have been subjected to saturating molecular screens in the genomic era. Many novel principles of development and cell biology owe their origins to this remarkably resilient frog.

Microinjection of Xenopus Oocytes.

Microinjection of Xenopus oocytes has proven to be a valuable tool in a broad array of studies that require expression of DNA or RNA into functional protein. These studies are diverse and range from expression cloning to receptor-ligand interaction to nuclear programming. Oocytes offer a number of advantages for such studies, including their large size (∼1.2 mm in diameter), capacity for translation, and enormous nucleus (0.3-0.4 mm). They are cost effective, easily manipulated, and can be injected in large numbers in a short time period. Oocytes have a large maternal stockpile of all the essential components for transcription and translation. Consequently, the investigator needs only to introduce by microinjection the specific DNA or RNA of interest for synthesis. Oocytes translate virtually any exogenous RNA regardless of source, and the translated proteins are folded, modified, and transported to the correct cellular locations. Here we present procedures for the efficient microinjection of oocytes and their subsequent care.

Identification of a Plasmodium falciparum inhibitor-2 motif involved in the binding and regulation activity of protein phosphatase type 1.

The regulation of Plasmodium falciparum protein phosphatase type 1 (PfPP1) activity remains to be deciphered. Data from homologous eukaryotic type 1 protein phosphatases (PP1) suggest that several protein regulators should be involved in this essential process. One such regulator, named PfI2 based on its primary sequence homology with eukaryotic inhibitor 2 (I2), was recently shown to be able to interact with PfPP1 and to inhibit its phosphatase activity, mainly through the canonical 'RVxF' binding motif. The details of the structural and functional characteristics of this interaction are investigated here. Using NMR spectroscopy, a second site of interaction is suggested to reside between residues D94 and T117 and contains the 'FxxR/KxR/K' binding motif present in other I2 proteins. This site seems to play in concert/synergy with the 'RVxF' motif to bind PP1, because only mutations in both motifs were able to abolish this interaction completely. However, regarding the structure/function relationship, mutation of either the 'RVxF' or 'FxxR/KxR/K' motif is more drastic, because each mutation prevents the capacity of PfI2 to trigger germinal vesicle breakdown in microinjected Xenopus oocytes. This indicates that the tight association of the PfI2 regulator to PP1, mediated by a two-site interaction, is necessary to exert its function. Based on these results, the use of a peptide derived from the 'FxxR/KxR/K' PfI2 motif was investigated for its potential effect on Plasmodium growth. This peptide, fused at its N-terminus to a penetrating sequence, was shown to accumulate specifically in infected erythrocytes and to have an antiplasmodial effect.

Extraction and nonradioactive detection of small RNA molecules.

The emergence of small RNAs as key and potent regulators of gene expression has prompted the need for robust detection and assay protocols to be developed for investigating their generation and tissue distribution. The physicochemical nature of these RNAs allows traditional assay methods to be employed; however, due to the relatively small size of endo-siRNAs, key changes to these protocols are required. Here, we present a method for the nonradioactive detection of endo-siRNAs in mouse tissue and microinjected Xenopus oocytes. The method comprises steps for RNA extraction, PAGE, and low-stringency northern blotting using DIG-labelled RNA probes. Moreover, it includes a strategy to design and generate cheap hybridization probes with greatly increased sensitivity. These methods may be used as a simple and robust protocol for nonradioactive detection of small RNAs or be combined with other strategies to potentially enhance signal intensity.

Analysis of Three-dimensional Systems for Developing and Mature Kidneys Clarifies the Role of OAT1 and OAT3 in Antiviral Handling

The organic anion transporters OAT1 (SLC22A6, originally identified by us as NKT) and OAT3 (SLC22A8) are critical for handling many toxins, metabolites, and drugs, including antivirals (Truong, D. M., Kaler, G., Khandelwal, A., Swaan, P. W., and Nigam, S. K. (2008) J. Biol. Chem. 283, 8654-8663). Although microinjected Xenopus oocytes and/or transfected cells indicate overlapping specificities, the individual contributions of these transporters in the three-dimensional context of the tissues in which they normally function remain unclear. Here, handling of HIV antivirals (stavudine, tenofovir, lamivudine, acyclovir, and zidovudine) was analyzed with three-dimensional ex vivo functional assays using knock-out tissue. To investigate the contribution of OAT1 and OAT3 in various nephron segments, the OAT-selective fluorescent tracer substrates 5-carboxyfluorescein and 6-carboxyfluorescein were used. Although OAT1 function (uptake in oat3(-/-) tissue) was confined to portions of the cortex, consistent with a proximal tubular localization, OAT3 function (uptake in oat1(-/-) tissue) was apparent throughout the cortex, indicating localization in the distal as well as proximal nephron. This functional localization indicates a complex three-dimensional context, which needs to be considered for metabolites, toxins, and drugs (e.g. antivirals) handled by both transporters. These results also raise the possibility of functional differences in the relative importance of OAT1 and OAT3 in antiviral handling in developing and mature tissue. Because the HIV antivirals are used in pregnant women, the results may also help in understanding how these drugs are handled by developing organs.

Microinjection of RNA and Preparation of Secreted Proteins from Xenopus Oocytes

Microinjection of Xenopus oocytes or embryos with messenger RNA (mRNA) is a powerful technique for studying development, including protein expression during development. This protocol describes a method for the preparation of secreted (35)S-labeled proteins following mRNA microinjection into Xenopus oocytes.

MRNA nuclear export at a glance

Eukaryotic gene expression is controlled by multiple mechanisms, and its regulation is central for physiological responses to extracellular and intracellular signals. An essential step in this process involves the movement of mRNA transcripts from the site of synthesis in the nucleus to the

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Panté, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus.Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

RAP55, a Cytoplasmic mRNP Component, Represses Translation in Xenopus Oocytes

mRNAs in eukaryotic cells are presumed to always associate with a set of proteins to form mRNPs. In Xenopus oocytes, a large pool of maternal mRNAs is masked from the translational apparatus as storage mRNPs. Here we identified Xenopus RAP55 (xRAP55) as a component of RNPs that associate with FRGY2, the principal component of maternal mRNPs. RAP55 is a member of the Scd6 or Lsm14 family. RAP55 localized to cytoplasmic foci in Xenopus oocytes and the processing bodies (P-bodies) in cultured human cells: in the latter cells, RAP55 is an essential constituent of the P-bodies. We isolated xRAP55-containing complexes from Xenopus oocytes and identified xRAP55-associated proteins, including a DEAD-box protein, Xp54, and a protein arginine methyltransferase, PRMT1. Recombinant xRAP55 repressed translation, together with Xp54, in an in vitro translation system. In addition, xRAP55 repressed translation in oocytes when tethered with a reporter mRNA. Domain analyses revealed that the N-terminal region of RAP55, including the Lsm domain, is important for the localization to P-bodies and translational repression. Taken together, our results suggest that xRAP55 is involved in translational repression of mRNA as a component of storage mRNPs.

Differential Stability of Biogenesis Intermediates Reveals a Common Pathway for Aquaporin-1 Topological Maturation

Topological studies of multi-spanning membrane proteins commonly use sequentially truncated proteins fused to a C-terminal translocation reporter to deduce transmembrane (TM) segment orientation and key biogenesis events. Because these truncated proteins represent an incomplete stage of synthesis, they transiently populate intermediate folding states that may or may not reflect topology of the mature protein. For example, in Xenopus oocytes, the aquaporin-1 (AQP1) water channel is cotranslationally directed into a four membrane-spanning intermediate, which matures into the six membrane-spanning topology at a late stage of synthesis (Skach, W. R., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., and Verkman, A. S. (1994) J. Cell Biol. 125, 803-815 and Lu, Y., Turnbull, I. R., Bragin, A., Carveth, K., Verkman, A. S., and Skach, W. R. (2000) Mol. Biol. Cell 11, 2973-2985). The hallmark of this process is that TM3 initially acquires an Nexo/Ccyto (Type I) topology and must rotate 180 degrees to acquire its mature orientation. In contrast, recent studies in HEK-293 cells have suggested that TM3 acquires its mature topology cotranslationally without the need for reorientation (Dohke, Y., and Turner, R. J. (2002) J. Biol. Chem. 277, 15215-15219). Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.

Compartment-specific phosphorylation of rat thyroid hormone receptor α1 regulates nuclear localization and retention

The thyroid hormone receptor α1 (TRα1) is a transcription factor, which can activate or repress gene expression in response to thyroid hormone. In addition, some of its actions, including DNA binding and transcriptional activation, are thought to be regulated by phosphorylation. Results presented here, using Xenopus oocyte microinjection assays, demonstrate that a phosphorylated form of rat TRα1 is present in the nucleus, whereas unphosphorylated TRα1 remains cytoplasmic. Changes in the phosphorylation state of TRα1 occur rapidly and point to the possibility that phosphorylation occurs in the nucleus. Furthermore, increasing the overall phosphorylation state of the cell leads to enhanced nuclear retention of TRα1, suggesting that compartment-specific phosphorylation regulates nuclear localization of TRα1. Enhanced nuclear retention of TRα1 is not dependent on phosphorylation of serine 12, a well-characterized casein kinase II site, nor is phosphorylation of this site necessary for import of TRα1 into the Xenopus oocyte nucleus. Similarly, mutational analysis in mammalian cells shows that nuclear localization and partitioning of TRα1 to the nuclear matrix are independent of serine 12 phosphorylation. Taken together, these studies suggest that phosphorylation of one or more sites in TRα1, excluding serine 12, enhances nuclear retention and/or inhibits nuclear export but is not directly involved in nuclear import.

Mutation or deletion of the C-terminal tail affects the function and structure of Xenopus laevis small heat shock protein, hsp30

Small heat shock proteins (shsps) act as molecular chaperones by preventing heat-induced aggregation and unfolding of cellular proteins by a mechanism that is still unclear. Previously we found that the C-terminal end of Xenopus shsp, hsp30C (30C), was essential for optimal chaperone activity. Examination of the C-terminal tail of 30C revealed that it had a net negative charge. Involvement of this negative charge in chaperone activity was assessed by the creation of two mutants, D209G (Asp converted to the more neutrally charged and less polar Gly at position 209) and D209/213G (Asp to Gly at position 209 and 213). Compared to 30C and D209G, D209/213G was impaired in inhibiting heat-induced citrate synthase aggregation. In rabbit reticulocyte lysate and Xenopus oocyte microinjection refolding assays the mutants were not as efficient as 30C in maintaining heat-treated luciferase in a folding competent state. Circular dichroism analysis revealed that D209G was similar in secondary structure to 30C whereas D209/213G displayed a loss of α-helical-like and β-sheet structure. Also, C-terminal truncation of 30C or 30D (an hsp30 isoform) resulted in a loss of secondary structure and function. This study clearly shows that mutation of aspartic acid residues in the C-terminal end of hsp30 or its truncation disrupts secondary structure and impairs its chaperone activity.

The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene.

Going back to the late 1970s and early 1980s, we trace the Xenopus oocyte microinjection experiments that led to the emergence of the concept of "modulator". The finding that the modulator could transactivate transcription from far upstream and in either orientation suggested that a new genetic element, different from the classical prokaryotic promoter sequences, had been discovered. This particular enhancer transactivates transcription of the sea urchin early (alpha) histone H2A gene which is regulated in early sea urchin development. We summarise the data from sea urchin microinjection experiments that confirm and extend the results obtained with Xenopus oocytes. We conclude that the H2A enhancer is bipartite, is located approx. 100 bp upstream of the TATAAATA box in the H2A gene of two sea urchin species and enhances transcription when placed at a position far upstream or far downstream of the gene unless an insulator intervenes between enhancer and promoter. Evidence from microinjection experiments with sea urchin embryos suggests that the developmental control of H2A expression resides not with the enhancer, which is constitutively active, but with a striking chromatin structure with two positioned nucleosomes near the 3' end of the gene. Within this structure, there is an insulator element.

Characterization and Regulation of a Cloned Ovine Gastrointestinal Peptide Transporter (oPepT1) Expressed in a Mammalian Cell Line

To investigate the kinetics of peptide transport by the peptide transporter, PepT1, Chinese hamster ovary cells were transfected with an expression vector containing our cloned ovine PepT1 cDNA. Transport was assessed by uptake studies using the radiolabeled dipeptide, [(3)H]-Gly-Sar. Expression of oPepT1 was detected at 8-24 h post-transfection with an optimal time of 16-24 h. Uptake of Gly-Sar by oPepT1 was pH-dependent with an optimal pH of 5.5-6.0, concentration-dependent and saturable with an apparent K(m) value of 1.0 +/- 0.1 mmol/L and a maximum velocity of 14.3 +/- 0.4 nmol/(mg protein x 40 min). Competition studies with nonradiolabeled peptides and [(3)H]-Gly-Sar showed that all di- and tripeptides inhibited uptake of [(3)H]-Gly-Sar. In addition, three tetrapeptides (Met-Gly-Met-Met, Pro-Phe-Gly-Lys, and Val-Gly-Ser-Glu) also inhibited [(3)H]-Gly-Sar uptake. There was no inhibition of [(3)H]-Gly-Sar uptake detected in the presence of nonradiolabeled free amino acids. Treatment of the cells with staurosporine, an inhibitor of protein kinase C (PKC) significantly increased the transport system. This increase was specific and could be blocked if treatment was done in the presence of phorbol 12-myristate-13-acetate (PMA), an activator of PKC. The staurosporine- and PMA-induced changes in peptide transport activity were not affected by cotreatment with cycloheximide. These data demonstrate that the transport of peptide substrates by oPepT1 in transfected mammalian cells is similar to that in microinjected Xenopus oocytes and that PKC phosphorylation plays a regulatory role in oPepT1 function.

Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state.

In this study we characterized the chaperone functions of Xenopus recombinant Hsp30C and Hsp30D by using an in vitro rabbit reticulocyte lysate (RRL) refolding assay system as well as a novel in vivo Xenopus oocyte microinjection assay. Whereas heat- or chemically denaturated luciferase (LUC) did not regain significant enzyme activity when added to RRL or microinjected into Xenopus oocytes, compared with native LUC, denaturation of LUC in the presence of Hsp30C resulted in a reactivation of enzyme activity up to 80-100%. Recombinant Hsp30D, which differs from Hsp30C by 19 amino acids, was not as effective as its isoform in preventing LUC aggregation or maintaining it in a folding-competent state. Removal of the first 17 amino acids from the N-terminal region of Hsp30C had little effect on its ability to maintain LUC in a folding-competent state. However, deletion of the last 25 residues from the C-terminal end dramatically reduced Hsp30C chaperone activity. Coimmunoprecipitation and immunoblot analyses revealed that Hsp30C remained associated with heat-denatured LUC during incubation in reticulocyte lysate and that the C-terminal mutant exhibited reduced affinity for unfolded LUC. Finally, we found that Hsc70 present in RRL interacted only with heat-denatured LUC bound to Hsp30C. These findings demonstrate that Xenopus Hsp30 can maintain denatured target protein in a folding-competent state and that the C-terminal end is involved in this function.

Splicing Factors SRp20 and 9G8 Promote the Nucleocytoplasmic Export of mRNA

We have uncovered a novel function for two members of the SR protein family in mRNA export. Using UV cross-linking, transient transfection, and Xenopus oocyte microinjection, we find that the nucleocytoplasmic shuttling proteins SRp20 and 9G8 interact specifically with a 22-nt RNA element from the histone H2a gene to promote the export of intronless RNAs in both mammalian cells and Xenopus oocytes. Antibodies to SRp20 or 9G8 eliminate RNA binding and significantly inhibit the export of RNAs carrying the element from oocyte nuclei. Our observation that SRp20 and 9G8 can be UV cross-linked to polyadenylated RNA in both the nucleus and cytoplasm of HeLa cells suggests a more general role for these SR proteins in mRNA export.

Nucleocytoplasmic shuttling of the thyroid hormone receptor alpha.

The thyroid hormone receptor alpha (TR alpha) exhibits a dual role as an activator or repressor of gene transcription in response to thyroid hormone (T(3)). Our studies show that TR alpha, formerly thought to reside solely in the nucleus tightly bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. The finding that TR alpha shuttles reveals an additional checkpoint in receptor control of gene expression. Using Xenopus oocyte microinjection assays, we show that there are two coexisting mechanisms for nuclear entry of TR alpha. First, nuclear import of TR alpha (molecular mass 46 kDa) was not sensitive to general inhibitors of signal-mediated transport, indicating that TR alpha can enter the oocyte nucleus by passive diffusion. Second, when TR alpha was tagged with glutathione-S:-transferase, import of the fusion protein (molecular mass 73 kDa) was completely blocked by these inhibitors, demonstrating that an alternative, signal-mediated import pathway exists for TR alpha. Nuclear retention of TR alpha in oocytes is enhanced in the presence of T(3), suggesting that more intranuclear binding sites are available for the ligand-bound receptor. Using mammalian cells, we show that shuttling of green fluorescent protein (GFP)-tagged and untagged TR alpha is inhibited in both chilled and energy-depleted cells, suggesting that there is an energy-requiring step in the nuclear retention/export process. Nuclear export of TR alpha is not blocked by leptomycin B, a specific inhibitor of the export receptor CRM1, indicating that TR alpha does not require the CRM1 pathway to exit the nucleus. Dominant negative mutants of TR with defects in DNA binding and transactivation accumulate in the cytoplasm at steady state, illustrating that even single amino acid changes in functional domains may alter the subcellular distribution of TR. In contrast to TR alpha, nuclear export of its oncogenic homolog v-ErbA is sensitive to leptomycin B, suggesting that the oncoprotein follows a CRM1-mediated export pathway. Acquisition of altered nuclear export capabilities may contribute to the oncogenic properties of v-ErbA.

Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

Chronic Ethanol Exposure Enhances Signaling Through Muscarinic Receptors Expressed by cRNA Injection in Xenopus Oocytes: Implications for Mechanism of Action

The molecular mechanisms underlying the cerebral symptoms of ethanol withdrawal syndrome are poorly understood. In addition to ethanol's effect on GABA and NMDA receptors, ethanol affects muscarinic acetylcholine signaling. This interaction has attracted attention because of the importance of muscarinic signaling in consciousness. Chronic ethanol exposure increases muscarinic receptor binding. Increased transcription of receptor message has been suggested as the underlying mechanism, but this hypothesis has not been tested directly. Therefore, we studied the effects of ethanol on muscarinic signaling in a model that bypasses transcription of muscarinic receptor genes. We expressed rat m1 muscarinic receptors by cRNA microinjection in Xenopus oocytes. Cells were voltage-clamped at -70 mV and effects of prolonged (24, 48, and 72 hr) exposure to ethanol (25, 50, and 100 mM) on methylcholine-induced calcium-activated Cl- currents were determined. Effects of prolonged ethanol exposure on currents induced by stimulation of lysophosphatidate receptors, direct G protein activation, or inositol trisphosphate receptor activation were studied as well. Prolonged ethanol exposure enhanced methylcholine (or lysophosphatidate-)-induced currents in a time- and concentration-dependent manner. Thus, enhanced muscarinic gene transcription is not required for ethanol enhancement of muscarinic signaling. Lack of ethanol effect on inositol trisphosphate-induced signaling suggests that intracellular signaling systems downstream of phospholipase C are not involved. In contrast, currents induced by direct G protein stimulation were enhanced significantly. Therefore, one potential site of ethanol's action on muscarinic signaling is upregulation of the associated G protein or enhancement of its functioning.