Target Cell Membrane Research Articles

Engineered Liposomes Protect Immortalized Immune Cells from Cytolysins Secreted by Group A and Group G Streptococci.

The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate the host cell plasma membrane. Recently developed liposomal nanotraps, mimicking the outer layer of the targeted cell membranes, serve as decoys for exotoxins, thus diverting them from attacking host cells. In this study, we develop a liposomal nanotrap formulation that is capable of protecting immortalized immune cells from the whole palette of cytolysins secreted by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis—important human pathogens that can cause life-threatening bacteremia. We show that the mixture of cholesterol-containing liposomes with liposomes composed exclusively of phospholipids is protective against the combined action of all streptococcal exotoxins. Our findings pave the way for further development of liposomal antivirulence therapy in order to provide more efficient treatment of bacterial infections, including those caused by antibiotic resistant pathogens.

Inflammation in Focus: The Beginning and the End.

The inflammation is an important biological response induced by various harmful stimuli, like viruses, bacterial infections, toxins, toxic compounds, tissue injury. During inflammation inflammatory cytokines and reactive oxygen species are produced. Inflammatory cytokines act on various receptors present on the plasma membrane of target cells. To initiate signaling cascade, and activate transcription factors, receptors should be internalized and enter the early endosomes, where the members of the signaling cascade can meet. The further cytoplasmic fate of the receptor plays crucial role in the progression and the course of inflammation. Usually acute inflammation removes injurious stimuli and helps to regain the normal healthy status of the organism. In contrast to this the uncontrolled chronic inflammation—stimulating other than immune cells, inducing transdifferentiation—can provide base of various serious diseases. This paper draws the attention of the long-lasting consequence of chronic inflammation, pointing out that one of the most important step in medication is to identify in time the factors initiating and maintaining inflammation.

Membrane fusion boosting drug transmembrane delivery

Benefiting from fusion element (peptides or proteins) anchoring, several biological membrane vesicles or synthetic liposomes have great potency to fuse with the target cell membrane via virus-mimetic behavior. When this process happens, the encapsulated cargos can be released directly into the cytoplasm together with membrane component (protein receptor, channel proteins, chemical receptors, antibodies) transfer, a process that facilitates cellular/molecular diagnosis and therapy.

Production of CD87 Antigen-Specific CAR-T Lymphocytes and Assessment of Their In Vitro Functional Activity

Aim. To generate anti-CD87 CAR-T lymphocytes and to assess their in vitro functional activity. Materials & Methods. Т-lymphocytes isolated from healthy donor peripheral blood were transduced with the anti-CD87-CAR, T2A, and FusionRed gene coding lentiviral vector. Transduction efficacy assessed by reporter protein FusionRed signal, subpopulation structure, and functional status of CAR-T lymphocytes were determined by flow cytometry. Interferon-y (IFN-y) expression by CAR-T lymphocytes was analyzed using immunoassay. Cytotoxic activity of CAR-T lymphocytes was evaluated during their co-cultivation with HeLa target cells by means of xCELLigence real-time assay. Results. The efficacy of T-lymphocyte transduction was 8.4 %. The obtained CAR-T cells contained the markers of both CD27 and/or CD28 activation (92.91 % cases) and PD1 exhaustion (20.66 % cases). The population of CAR-T lymphocytes showed 98.51 % central memory T-cell phenotype and CD4/CD8 ratio of 1:7. IFN-y concentration in the medium after co-cultivation of CAR-T lymphocytes with target cells appeared to be significantly higher than in control samples. The study demonstrates that generated CAR-T lymphocytes manifest specific cytotoxicity towards target cells with both unmodified expression and overexpression of CD87 antigen in HeLa cell lines. Cytotoxicity proved to be more pronounced with respect to the cell line with CD87 antigen overexpression. Conclusion. Despite overexpression of PD1 exhaustion marker, CAR-T lymphocytes showed specific IFN-y secretion and pronounced cytotoxic activity in interaction with CD87 antigen on target cell membranes. Therefore, anti-CD87 CAR-T lymphocytes can be applied in the treatment of hematologic as well as solid tumors. Since the observed difference in cytotoxicity does not linearly correlate with CD87 antigen density on the surface of attacked cells, the in vivo administration of a CAR-T cell drug should be designed to prevent cytotoxic risk for CD87-expressing healthy cells.

Novel structural insights for a pair of monoclonal antibodies recognizing non-overlapping epitopes of the glucosyltransferase domain of Clostridium difficile toxin B.

Clostridium difficile toxins are the primary causative agents for hospital-acquired diarrhea and pseudomembranous colitis. Numerous monoclonal antibodies (mAbs) targeting different domains of Clostridium difficile toxin have been reported. Here we report the crystal structures of two mAbs, B1 and B2, in complex with the glycosyltransferase domain (GTD) of the Clostridium difficile toxin B (TcdB). B2 bound to the N-terminal 4 helix bundle of the GTD, a conserved membrane localization domain (MLD) found in the large clostridial glycosylating toxin family implicated in targeting plasma membrane. B1 bound to a distinct epitope at the hinge region between the MLD and the catalytic subdomain of the GTD. Functional studies revealed the potency of these mAbs in vitro and in vivo to be synergistic when given in combination.

New approaches related to the use of polyamine-free and polyamine-deficient diets in the list of nutritional products for COVID-19 patients

Researches aimed at finding effective means of pathogenic therapy for this viral infection are extremely relevant. Researches of the last three years have established that some human pathogenic coronaviruses – MERS, SARS-CoV and SARS-CoV-2, contain aliphatic polyamines in their structure, which participate in the packaging of genetic material (DNA, RNA), as well as the nucleocapsid. Virus-host cell interactions also provide adhesion processes on the surface of the cytoplasmic membrane of target cells. In the intracellular space, aliphatic polyamines actively affect the translation and replication processes of the genetic material and necessary proteins of the virus itself, as well as the formation of daughter viruses. Long-term persistence in the SARS-CoV-2 infected organism is largely due to the absorption of polyamines by coronavirus localized in target cells of the blood and parenchymatous organs. Unfortunately, the above new facts did not serve as a prerequisite for finding effective means of pathogenetic therapy for COVID-19, based on the inhibition of polyamine-dependent processes that ensure long-term persistence of SARS-CoV-2 in the infected organism. We are talking about specific drugs such as α-difluoromethylornithine and its analogues, which are successfully used in oncology in the complex treatment of malignant neoplasms with the aim of lowering the level of aliphatic polyamines in the affected areas of malignantly transformed organs. We recommend the use of polyamine-free and polyamine-deficient diets for COVID-19 for the first time. In the planned study, we will present tables with food products of animal and vegetable origin, characterized by extremely low content and/or absence of aliphatic polyamines in them. At the same time, food products with a high content of aliphatic polyamines should be excluded from the general list of products recommended for COVID-19 patients. We also recommend the use of a polyamine-deficient diet (with a preventive purpose) during the COVID-19 pandemic to a wide contingent of practically healthy individuals, convalescents, medical staff of specialized infectious disease clinics, as well as family members of SARS-CoV-2 infected patients.

Novel Indole Alkaloids from Rauvolfia Yunnanensis, Inhibiting Esbl Producing Escherichia Coli by Targeting Cell Membrane Integrity

Novel Indole Alkaloids from Rauvolfia Yunnanensis, Inhibiting Esbl Producing Escherichia Coli by Targeting Cell Membrane Integrity

Cancer cell membrane targeting and red light-triggered carbon monoxide (CO) release for enhanced chemotherapy.

Chemotherapy assisted by carbon monoxide (CO) gas therapy is an emerging powerful cancer therapeutic modality. However, the effective delivery and controlled release of CO in tumor cells remain a challenge. Herein, a cell membrane bionic nano delivery system (RBC-H@DOX/3-HF@MSN, termed as RHM) was designed to selectively accumulate in tumors and generate CO in situ upon red light irradiation for the combination of chemotherapy and gas therapy. CO significantly improves the therapeutic effect of DOX from 29.0% to 82.4%.

Cell-surface glycans act as attachment factors for porcine hemagglutinating encephalomyelitis virus

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a neurotropic coronavirus and highly pathogenic in veterinary clinic. Spike (S) protein of PHEV interplays with host components to cross the plasma membrane of target cells, but characterization of its functional receptors is limited. Here, we discovered that cell-surface glycans, i.e., sialic acid (SA) and heparan sulfate (HS), act as critical interacting factors of PHEV, involving in viral attachment. As shown in glycans depletion assay, removing SA or HS from N2a cells inhibits PHEV infection. Soluble sugar monomers were utilized for competitive binding tests, and we found that both SA and HS could specifically bind to PHEV and affect the viral infectivity. Furthermore, the expression of heparan sulfate proteoglycans (HSPGs), including syndecans and glypicans, and endoglycosidase heparinase which cleaves HS were regulated by PHEV RNA replication. Together, we newly identified specificity recognition of cellular glycans and PHEV during infection, providing novel cellular targets for antiviral therapies and better understanding of pathogenesis.

Profiling Cancer Cells by Cell-SELEX: Use of Aptamers for Discovery of Actionable Biomarkers and Therapeutic Applications Thereof.

The identification of tumor cell-specific surface markers is a key step towards personalized cancer medicine, allowing early assessment and accurate diagnosis, and development of efficacious targeted therapies. Despite significant efforts, currently the spectrum of cell membrane targets associated with approved treatments is still limited, causing an inability to treat a large number of cancers. What mainly limits the number of ideal clinical biomarkers is the high complexity and heterogeneity of several human cancers and still-limited methods for molecular profiling of specific cancer types. Thanks to the simplicity, versatility and effectiveness of its application, cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology is a valid complement to the present strategies for biomarkers’ discovery. We and other researchers worldwide are attempting to apply cell-SELEX to the generation of oligonucleotide aptamers as tools for both identifying new cancer biomarkers and targeting them by innovative therapeutic strategies. In this review, we discuss the potential of cell-SELEX for increasing the currently limited repertoire of actionable cancer cell-surface biomarkers and focus on the use of the selected aptamers as components of innovative conjugates and nano-formulations for cancer therapy.

Epigenetic modifications of tumor necrosis factor-alpha in joint cartilage tissue from osteoarthritis patients - CONSORT.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

Peptide-based Fusion Inhibitors for Preventing the Six-helix Bundle Formation of Class I Fusion Proteins: HIV and Beyond.

A number of different viral families have developed convergent methods to infect cells. Class I fusion proteins are commonly used by members of Arenaviridae, Coronaviridae, Filovirdae, Orthomyxoviridae, Paramyxoviridae, and Retroviridae. Class I viral fusion proteins are trimers that are involved in recognizing the cellular receptor, with a region that is responsible for fusing the viral and target cell membranes. During the fusion process, the fusion region folds into a six-helix bundle (6 HB) which approximates the two membranes leading to fusion. For Human Immunodeficiency Virus (HIV), the gp41 subunit is responsible for the formation of this 6 HB. The fusion inhibitor drug enfuvirtide, or T20, is the only US Food and Drug Administration and European Medicines Agency approved drug which targets this crucial step and has been widely used in combination regimens for the treatment of HIV since March 2003. In this review, we describe the current state of peptide-based fusion inhibitors in the treatment of HIV, and review how the field of HIV research is driving advances in the development of similar therapeutics in other viral systems, including the Severe Acute Respiratory Syndrome (SARS) coronaviruses.

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

ABSTRACT Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell–cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

Structural and functional insights into colicin: a new paradigm in drug discovery.

Colicins are agents of allelopathic interactions produced by certain enterobacteria which give them a competitive advantage in the environment. These protein molecules are mostly encoded by plasmids. The colicin operon consists of the activity, immunity and the lysis genes. The activity protein is responsible for the killing activity, the immunity protein protects the producer cell from the lethal action of colicin and the lysis protein facilitates its release. Colicins are primarily composed of three domains, namely the receptor-binding domain, the translocation domain and the cytotoxic domain. The protein molecule binds to its cognate receptor on the target cell via the receptor-binding domain and undergoes translocation into the cell either via the Tol system or the Ton system. After gaining entry into the target cell, there are various mechanisms by which colicins exert their lethality. These comprise DNase activity, RNase activity and pore formation in the target cell membrane or peptidoglycan synthesis inhibition. This review gives a detailed insight into the structural and functional aspect of colicins and their mode of action. This knowledge is of immense significance because colicins are being considered as very useful alternatives to conventional antibiotics in the treatment of multidrug-resistant infections. Besides, they also have a negligible harmful impact on the commensals. Thus, before tapping their therapeutic potential, it is imperative to know their structure and mechanism of action in detail.

Antibacterial mechanisms of thyme essential oil nanoemulsions against Escherichia coli O157:H7 and Staphylococcus aureus: Alterations in membrane compositions and characteristics

In this study, the antibacterial activities of thyme essential oil nanoemulsions (TEON) prepared by ultrasonication (US) and high-pressure homogenization (HPH) were evaluated via minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-killing curve analyses. TEON-US presented superior bacteriostatic and bactericidal effects, which was found to be correlated with its better physicochemical characteristics (including smaller droplet size and higher zeta potential) compared to TEON-HPH. The underlying antibacterial mechanisms were specifically investigated targeting cell membranes of Escherichia coli O157:H7 (Gram-negative bacterium) and Staphylococcus aureus (Gram-positive bacterium). Thereinto, membrane disintegrations were observed followed by massive leakage of cytoplasmic inclusions. Furthermore, the membrane dysfunctions (involving membrane potential depolarization and fluidity reduction) were likely associated with the modifications seen in the fatty acid composition upon TEONs exposure, and ultimately causing bacterial death. These results deciphered the mode of antibacterial action of TEONs, providing a critical theoretical basis for their future application in food matrix.

Novel Bent Conformation of CD4 Induced by HIV-1 Inhibitor Indirectly Prevents Productive Viral Attachment

GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen–deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4′s flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.

Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID‐19 Therapy (Adv. Mater. 49/2021)

COVID-19 Therapy In their work reported in article number 2103471, Long Zhang, Fangfang Zhou, and co-workers fuse the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with angiotensin converting enzyme 2 (ACE2) and engineer extracellular vesicles (EVs) on their surface enriched with palmitoylated ACE2 (PM-ACE2-EVs). The PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell-surface ACE2, thereby preventing SARS-CoV-2 from entering the host cell. This study provides a novel EV-based candidate for prophylactic and therapeutic treatment against COVID-19.

Antibacterial activity of Sapindus saponins against microorganisms related to food hygiene and the synergistic action mode of Sapindoside A and B against Micrococcus luteus in vitro

Sapindus saponins are natural nonionic surfactants, and they have a broad-spectrum antibacterial effect, but few studies have elucidated the antibacterial mechanism of Sapindus saponins to date. Therefore, this study screened the antibacterial activity of Sapindus saponins singly and in combination against 7 bacteria, and investigated the synergistic antibacterial action via targeting cell membrane proteins. The only combination of Sapindoside A and B (S AB ) with synergistic activity against Micrococcusluteus ( M. luteus ) was obtained, where the MICs of Sapindoside A and B were one fourth of their individual minimum inhibitory concentration (MIC). After treatment with S AB , the spectral features of protein in the cell membrane of M. luteus showed obvious changes based on Raman spectroscopy, and the constitutions of membrane proteins were changed seriously. Besides, changes in protein microenvironment was observed, and the contribution of Sapindoside A was greater than that of Sapindoside B with concentration below 2MIC. Molecular docking also demonstrated that Sapindoside A interacted with penicillin-binding protein 2, and showed higher binding energy than Sapindoside B, further indicating that the greater contribution in the synergistic action of S AB on membrane proteins. Collectively, these results showed that the synergistic antibacterial action of Sapindoside A and B against M. luteus could be achieved by attacking cell membrane proteins, and Sapindoside A played a major role, suggesting that S AB has the potential to be the natural antibacterial detergent additive in food industry, although it is limited in its scope of antimicrobial activity against foodborne bacteria. • Synergistic antibacterial combination of Sapindoside A and B (S AB ) was obtained. • Spectral features of membrane protein changed greatly based on Raman spectroscopy. • Degradation of membrane proteins and change in protein microenvironment was observed. • Contribution of Sapindoside A was greater than that of Sapindoside B in the S AB .

Abstract 14182: Diverse microRNA Cargo is Essential for the Cardioprotective Effects of Therapeutic Extracellular Vesicles

Background: Extracellular vesicles (EVs) deliver their protein and RNA cargo across the plasma membrane of target cells to exert a functional response. Injection of cardiosphere-derived cell (CDC) EVs have been shown to reduce cardiomyocyte (CM) cell death and improve cardiac function in large and small animal models of myocardial infarction (MI) and ischemia-reperfusion. While several EV miRNAs (miRs) have been identified to play a role in cardioprotection, it has not yet been established whether miR cargo is necessary for the therapeutic benefit of EVs. We hypothesized that miRNA cargo is essential in CDC-EV mediated cardioprotection post-MI. Methods: CDCs were transfected with siRNA against Drosha, the initial endonuclease in the miR biogenesis pathway. EVs were isolated from serum-free media and characterized by size, morphology, and protein/miR expression as previously described. We examined the role of EV miRNA on target cell apoptosis, proliferation, angiogenesis, and gene expression using a series of in vitro assays. To determine if miR cargo is essential for the therapeutic efficacy of CDC-EVs in vivo , we induced acute MI in adult C57BL/6 mice and randomized them to receive 1) EVs, 2) miR depleted EVs or 3) vehicle control. LVEF was assessed by echo at 1- and 28-days post-MI and tissue samples processed for assessment of histological endpoints. Results: Transfection of CDCs with siRNA against Drosha reduces miRNA in CDCs and their EVs by 81±5% and 65±4%, respectively (mean ± SEM, t-test, p<0.001). miR depleted EVs failed to recapitulate the reduction in cardiomyocyte apoptosis, decrease in fibroblast proliferation, and enhancement in angiogenesis seen with control EVs. Furthermore, while intramyocardial administered CDC-derived EVs improved LVEF compared with vehicle control treated animals 28 days post-MI (62 ± 4% vs 44 ± 4%), mice treated with miR-depleted EVs demonstrated no significant difference in LVEF (42 ± 6%) compared with vehicle control (n=8 mice/group, mean ± SEM, p<0.05, one-way ANOVA). Conclusions: miR deficient human CDC-EVs are unable to recapitulate the cardioprotective effects seen with control extracellular vesicles, providing important insight into the mechanism of action of therapeutic EVs.

Subsynaptic Distribution, Lipid Raft Targeting and G Protein-Dependent Signalling of the Type 1 Cannabinoid Receptor in Synaptosomes from the Mouse Hippocampus and Frontal Cortex

Numerous studies have investigated the roles of the type 1 cannabinoid receptor (CB1) in glutamatergic and GABAergic neurons. Here, we used the cell-type-specific CB1 rescue model in mice to gain insight into the organizational principles of plasma membrane targeting and Gαi/o protein signalling of the CB1 receptor at excitatory and inhibitory terminals of the frontal cortex and hippocampus. By applying biochemical fractionation techniques and Western blot analyses to synaptosomal membranes, we explored the subsynaptic distribution (pre-, post-, and extra-synaptic) and CB1 receptor compartmentalization into lipid and non-lipid raft plasma membrane microdomains and the signalling properties. These data infer that the plasma membrane partitioning of the CB1 receptor and its functional coupling to Gαi/o proteins are not biased towards the cell type of CB1 receptor rescue. The extent of the canonical Gαi/o protein-dependent CB1 receptor signalling correlated with the abundance of CB1 receptor in the respective cell type (glutamatergic versus GABAergic neurons) both in frontal cortical and hippocampal synaptosomes. In summary, our results provide an updated view of the functional coupling of the CB1 receptor to Gαi/o proteins at excitatory and inhibitory terminals and substantiate the utility of the CB1 rescue model in studying endocannabinoid physiology at the subcellular level.