Albumin inhibits neutrophil spreading and hydrogen peroxide release by blocking the shedding of CD43 (sialophorin, leukosialin)

Spreading of neutrophils on protein-coated surfaces is a pivotal event in their ability to respond to soluble, physiologic agonists by releasing large amounts of hydrolases and oxidants. Using neutrophils plated on serum-, fibrinogen- or fibronectin-coated surfaces, we investigated the effect of human serum albumin (HSA) on spreading-dependent neutrophil responses. HSA suppressed the respiratory burst of neutrophils in response to tumor necrosis factor-alpha (TNF), complement component C5a or formylated peptide, but not phorbol myristate acetate. HSA was suppressive only if added before the onset of the respiratory burst, and suppression was reversed when HSA was removed. Likewise, HSA selectively and reversibly inhibited TNF-induced cell spreading and the associated fall in cAMP. However, HSA did not hinder TNF-induced cell adherence to the same protein-coated surfaces. We investigated cell surface sialoproteins as modulators of cell spreading and as targets for the anti-spreading action of HSA. Oxidation of the cell surface with periodate followed by reduction with 3H-borohydride and immunoblotting with specific mAbs helped identify the predominant sialoprotein on human neutrophils as CD43 (sialophorin, leukosialin). Treatment of neutrophils with C. perfringens sialidase desialylated CD43, markedly enhanced the ability of the cells to respond to TNF by spreading and undergoing a respiratory burst, and antagonized the ability of HSA to inhibit these responses. TNF-treated, adherent neutrophils shed CD43, and this was blocked by HSA, but not by ovalbumin. Exogenous neutrophil elastase removed CD43 from the neutrophil surface. HSA blocked the actions of both sialidase and elastase on CD43. In contrast, ovalbumin did not block the action of sialidase on CD43, and HSA did not inhibit the ability of sialidase to hydrolyze a synthetic substrate. These results suggested that HSA might bind CD43. In fact, the extracellular portion of CD43 bound to HSA-Sepharose, but not to ovalbumin- or glycylglycine-Sepharose. Finally, two mAbs recognizing different epitopes on CD43 mimicked HSA's inhibitory effects on neutrophil function. Thus, HSA can dissociate attachment of neutrophils from spreading. This dissociation may help neutrophils migrate along a chemotactic gradient, while decreasing their release of oxidants. CD43, a long, rigid molecule with a markedly negative charge, antagonizes neutrophil spreading. HSA appears to inhibit spreading-dependent neutrophil functions by binding to CD43 and interfering with the ability of neutrophils to shed it.     

Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain

The cytoplasmic face of the Golgi contains a variety of proteins with coiled-coil domains. We identified one such protein in a yeast two-hybrid screen, using as bait the peripheral Golgi phosphatidylinositol(4,5)P2 5-phosphatase OCRL1 that is implicated in a human disease, the oculocerebrorenal syndrome. The approximately 2.8-kilobase mRNA is ubiquitously expressed and abundant in testis; it encodes a 731-amino acid protein with a predicted mass of 83 kDa. Antibodies against the sequence detect a novel approximately 84-kDa Golgi protein we termed golgin-84. Golgin-84 is an integral membrane protein with a single transmembrane domain close to its C terminus. In vitro, the protein inserts post-translationally into microsomal membranes with an N-cytoplasmic and C-lumen orientation. Cross-linking indicates that golgin-84 forms dimers, consistent with the prediction of an approximately 400-residue dimerizing coiled-coil domain in its N terminus. The dimerization potential is supported by a data base search that showed that the N-terminal 497 residues of golgin-84 contain a coiled-coil domain that when fused to the RET tyrosine kinase domain had the ability to activate it, forming the RET-II oncogene. Data base searching also indicates golgin-84 is similar in structure and sequence to giantin, a membrane protein that tethers coatamer complex I vesicles to the Golgi.     

A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion

A human CD2 cytoplasmic tail-binding protein, termed CD2BP1, was identified by an interaction trap cloning method. Expression of CD2BP1 is restricted to hematopoietic tissue, being prominent in T and natural killer (NK) cells, with long (CD2BP1L) and short (CD2BP1S) variants arising by alternative RNA splicing. Both CD2BP1 molecules are homologous to Schizosaccharomyces pombe cdc15, and include a helical domain, variable length intervening PEST sequence and C-terminal SH3 domain. Although the CD2BP1 SH3 domain binds directly to the CD2 sequence, KGPPLPRPRV (amino acids 300-309), its association is augmented markedly by the CD2BP1 N-terminal segment. Upon ligand-induced clustering of surface CD2 molecules, CD2BP1 redistributes from a cytosolic to a surface membrane compartment, co-localizing with CD2. In turn, CD2-stimulated adhesion is downregulated by CD2BP1, apparently through coupling of the protein tyrosine phosphatase (PTP)-PEST to CD2. These findings offer the first molecular view into the control processes for T cell adhesion.     

Production of the thyrotrophin receptor extracellular domain as a glycosylphosphatidylinositol-anchored membrane protein and its interaction with thyrotrophin and autoantibodies

The thyrotrophin (TSH) receptor (TSHR) is synthesized as a single polypeptide with a predicted large extracellular domain (ECD), a seven-transmembrane pass region and a C-terminal intracellular tail. It is a common target for production of autoantibodies. To investigate whether the ECD is solely responsible for ligand interaction, we directed the expression of this domain in isolation on the cell surface by means of a glycosylphosphatidylinositol (GPI) anchor sequence. Immunoblotting detected TSHR material of Mr 70,000 expressed at high levels. In immunoprecipitation studies, the GPI-anchored ECD was recognized by experimental and pathological antibodies. The molecule was detected on the cell surface by flow cytofluorimetry at up to 10-fold higher amounts than the highest expressing full-length receptor clone. Radioligand binding studies confirmed this and showed that the recombinant molecule bound TSH with high affinity similar to full-length receptor; however, studies with human autoimmune sera indicated differences in the degree of inhibition when compared with full-length receptor. The existence of the GPI anchor was confirmed by cleavage with a GPI-specific phospholipase C and biosynthetic labeling with [3H]ethanolamine. TSHR material was also present inside the cell in both soluble and membrane-bound forms. Thus, the recombinant GPI-anchored ECD is the smallest known fragment of the TSHR that retains high-affinity TSH binding and is expressed at high levels on the cell surface as well as internally; this approach may well be useful for other membrane proteins.     

Growth-associated protein (GAP-43), its mRNA, and protein kinase C (PKC) isoenzymes in brain regions of depressed suicides

Thirty-three patients referred to a wasting clinic were evaluated to assess whether levels of HIV RNA were related to the magnitude of prior weight loss. Their median RNA level was 46,887 gene copies/ml (range, <200-510,070 gene copies/ml) at the time of referral. Patients had lost 10.5 +/- 6.4 kg over 461 +/- 304 days. RNA levels were correlated with the absolute amount and percentage of weight lost as well as the difference in body mass index (BMI) at the prior maximal and minimal recorded weights (r = 0.7, 0.67, 0.69; p = .0001 for the comparisons). The magnitude of these changes increased across strata of HIV RNA levels (p < or = .004), previously defined as associated with increasing risk for disease progression. The other parameter that could be associated with weight loss was the CD4 lymphocyte count (r = -0.43; p = .01). Low levels of testosterone and measures of body cell mass, fat free mass, or fat mass within 6 weeks of the RNA level could not be related to weight loss, change in BMI, or RNA levels. Thirty-two of the patients had chronic, relentless weight loss; in 15 of these subjects, no apparent secondary opportunistic complications were associated with weight loss or gastrointestinal symptoms to impair energy intake. Levels of HIV replication appear to be causally related to the magnitude of weight loss in some patients with wasting.     

Transdermal immunisation with an integral membrane component, gap junction protein, by means of ultradeformable drug carriers, transfersomes

Molecules greater than 500 Da normally do not cross the skin. This prevents epicutaneous delivery of the high molecular weight therapeutics as well as non-invasive transcutaneous immunisation. Extremely deformable vesicles prepared by the judicious combination of several materials provide a solution to this problem: the resulting agent carriers, transfersomes, are the only tested colloidal system that can transport even large macromolecules spontaneously through the skin in immunologically active form. Gap junction proteins (GJP) incorporated into transfersomes and applied to the intact skin surface thus give rise to specific antibody titres marginally higher than those elicited by subcutaneous injections of GJP in transfersomes, mixed lipid micelles or liposomes. The latter two carrier systems give no significant biological response after epicutaneous administration. Transcutaneous protein delivery by means of transfersomes also appears to increase the relative concentration of anti-GJP IgA in the serum.     

The protein translocation apparatus contributes to determining the topology of an integral membrane protein in Escherichia coli

The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus. We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli. In particular, we studied the effects of prlA mutations on membrane protein topology. These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus. We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants. We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology.     

A conserved sequence motif in the integrin beta3 cytoplasmic domain is required for its specific interaction with beta3-endonexin

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.     

BCMAp: an integral membrane protein in the Golgi apparatus of human mature B lymphocytes

BCMA is a human gene expressed preferentially in mature B lymphocytes as a 1.2 kb mRNA, which encodes a 184 amino acid peptide (BCMAp). The study of BCMA mRNA expression, using human malignant B cell lines characteristic of different stages of B lymphocyte differentiation, demonstrated that the BCMA mRNA is absent in the pro-B lymphocyte stage. It is expressed faintly at the pre-B cell stage and its expression increases with B lymphocyte maturation. Polyclonal antibodies were used to show, by cellular fractionation and immunoprecipitation, that BCMAp is a non-glycosylated integral membrane protein. Furthermore, BCMAp inserts, in vitro, into canine microsomes, as a type I integral membrane protein. Cell surface labeling showed that BCMAp is not expressed in the plasma membrane of mature B lymphocytes. Immunofluorescence studies revealed that BCMAp lies in a cap-like structure near the nucleus, that was identified as the Golgi apparatus by co-localization of BCMAp with CTR433, a marker of the medial cisternae of the Golgi apparatus. Confocal scanning laser microscopy of U266 plasma cells labeled with markers of various Golgi apparatus subcompartments strongly suggests that BCMAp is located in the cis part of the Golgi apparatus. Thus, BCMAp is the first Golgi resident protein with a tissue specificity and whose expression is linked to the stage of differentiation of B lymphocytes. The location of BCMAp in the Golgi apparatus and its high expression in plasmocytes (secreting large amounts of Ig) suggest that BCMAp is implicated in the intracellular traffic of Ig.     

Differential localization of alpha-actinin and the 30 kD actin-bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum

Dictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, alpha-actinin and the 30 kD actin-bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for alpha-actinin is not apparent in this region. By contrast, alpha-actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of alpha-actinin with the contractile vacuole was confirmed by colocalization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and alpha-actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but disassociates from phagosomes soon after internalization [Furukawa et al., 1992: Protoplasma 169: 18-27]. alpha-actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has disassociated. These results support the hypothesis that alpha-actinin and the 30 kD protein play distinct roles in cell structure and movement in Dictyostelium.     

Human immunodeficiency virus type 1 Vpu protein induces degradation of CD4 in vitro: the cytoplasmic domain of CD4 contributes to Vpu sensitivity

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus (HIV) receptor for infection of human host cells. We have recently demonstrated that Vpu, an HIV type 1 (HIV-1) encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. In this report, we describe an in vitro model system that allowed us to define important parameters for Vpu-dependent CD4 degradation. The rate of CD4 decay in rabbit reticulocyte lysate was approximately one-third of that observed previously in tissue culture experiments in the presence of Vpu (40 versus 12 min) and required no other HIV-1 encoded proteins. Degradation was contingent on the presence of microsomal membranes in the assay and the coexpression of Vpu and CD4 in the same membrane compartment. By using the in vitro degradation assay, the effects of specific mutations in CD4, including C-terminal truncations and glycosylation mutants, were analyzed. The results of these experiments indicate that Vpu has the capacity to induce degradation of glycosylated as well as nonglycosylated membrane-associated CD4. Truncation of 13 C-terminal amino acids of CD4 did not affect the ability of Vpu to induce its degradation. However, the removal of 32 amino acids from the C-terminus of CD4 completely abolished sensitivity to Vpu. This suggests that Vpu targets specific sequences in the cytoplasmic domain of CD4 to induce its degradation. We also analyzed the effects of mutations in Vpu on its biological activity in the in vitro CD4 degradation assay. The results of these experiments suggest that sequences critical for this function of Vpu are located in its hydrophilic C-terminal domain.     

A cluster of basic repeats in the dystrophin rod domain binds F-actin through an electrostatic interaction

The dystrophin rod domain is composed of 24 spectrin-like repeats and was thought to act mainly as a flexible spacer between the amino-terminal actin binding domain and carboxyl-terminal membrane-associated domains. We previously demonstrated that a fragment of the dystrophin rod domain also binds F-actin. However, the nature and extent of rod domain association with F-actin is presently unclear. To begin addressing these questions, we characterized two recombinant proteins representing adjacent regions of the dystrophin rod. DYS1416 (amino acids 1416-1880) bound F-actin with a Kd of 14.2 +/- 5.2 microM and a stoichiometry of 1 mol:mol of actin. However, DYS1030 (amino acids 1030-1494) failed to bind F-actin, suggesting that not all rod domain repeats are capable of binding F-actin. Interestingly, DYS1416 corresponds to a unique region of the dystrophin rod rich in basic amino acids, whereas DYS1030 is composed mainly of acidic repeats. This observation suggested that DYS1416 may interact with acidic actin filaments through an electrostatic interaction. Supporting this hypothesis, actin binding by DYS1416 was dramatically inhibited by increasing ionic strength. We suggest that electrostatic interactions between basic spectrin-like repeats and actin filaments may contribute to the actin binding activity of other members of the actin cross-linking protein family.     

Molecular characterization of GCP170, a 170-kDa protein associated with the cytoplasmic face of the Golgi membrane

We have isolated a cDNA clone encoding a protein (designated GCP170) of 1530 amino acid residues with a calculated molecular mass of 170 kDa that is localized to the Golgi complex. Hydropathy analysis shows that GCP170 contains no NH2-terminal signal sequence nor a hydrophobic domain sufficient for participating in membrane localization. It is also predicted that GCP170 has characteristic secondary structures including an extremely long alpha-helical domain that likely forms a coiled-coil between non-coil domains at the NH2 and COOH termini, suggesting that the protein is organized as a globular head, a stalk, and a tail. Immunocytochemical observations revealed that GCP170 was localized to the Golgi complex and the cytoplasm, consistent with biochemical data indicating that the protein exits as a membrane-associated form and a soluble form. GCP170 was dissociated from the Golgi membrane in response to brefeldin A as rapidly as a coat protein complex of non-clathrin-coated vesicles (beta-COP, a subunit of coatomer), but did not co-localize with beta-COP on the Golgi membrane when examined by immunoelectron microscopy. The protein was detected as phosphorylated and unphosphorylated forms, of which the unphosphorylated form was more tightly associated with the Golgi membrane. When cells were extracted with 1% Triton X-100 under microtubule-stabilizing conditions, GCP170 remained in the cells in association with the Golgi complex. These results indicate that GCP170 is a peripheral membrane protein with a long coiled-coil domain that may be involved in the structural organization or stabilization of the Golgi complex.     

Outer membrane protein B1, an iron-repressible protein conserved in the outer membrane of Moraxella (Branhamella) catarrhalis, binds human transferrin

Moraxella (Branhamella) catarrhalis is a gram-negative human mucosal pathogen, which primarily causes otitis media in young children. However, this bacterium is also a common cause of lower respiratory tract infections in adults with underlying lung disease. Our previous data have shown that M. catarrhalis expresses iron-repressible outer membrane proteins in response to iron limitation. We have extended these observations to demonstrate that one of these proteins, termed outer membrane protein (OMP) B1, binds human transferrin. Using a newly developed monoclonal antibody to OMP B1, we determined that this protein is conserved in the iron-stressed outer membranes of all clinical isolates of M. catarrhalis tested to date. Furthermore, our data have confirmed that children infected with M. catarrhalis have immunoglobulin G antibodies to OMP B1 in their convalescent sera. These current data suggest that OMP B1 is immunogenic and expressed in vivo and may be involved in an iron uptake mechanism utilized by M. catarrhalis.     

Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB

FhuD is the periplasmic binding protein of the ferric hydroxamate transport system of Escherichia coli. FhuD was isolated and purified as a His-tag-labeled derivative on a Ni-chelate resin. The dissociation constants for ferric hydroxamates were estimated from the concentration-dependent decrease in the intrinsic fluorescence intensity of His-tag-FhuD and were found to be 0.4 microM for ferric aerobactin, 1.0 microM for ferrichrome, 0.3 microM for ferric coprogen, and 5.4 microM for the antibiotic albomycin. Ferrichrome A, ferrioxamine B, and ferrioxamine E, which are poorly taken up via the Fhu system, displayed dissociation constants of 79, 36, and 42 microM, respectively. These are the first estimated dissociation constants reported for a binding protein of a microbial iron transport system. Mutants impaired in the interaction of ferric hydroxamates with FhuD were isolated. One mutated FhuD, with a W-to-L mutation at position 68 [FhuD(W68L)], differed from wild-type FhuD in transport activity in that ferric coprogen supported promotion of growth of the mutant on iron-limited medium, while ferrichrome was nearly inactive. The dissociation constants of ferric hydroxamates were higher for FhuD(W68L) than for wild-type FhuD and lower for ferric coprogen (2.2 microM) than for ferrichrome (156 microM). Another mutated FhuD, FhuD(A150S, P175L), showed a weak response to ferrichrome and albomycin and exhibited dissociation constants two- to threefold higher than that of wild-type FhuD. Interaction of FhuD with the cytoplasmic membrane transport protein FhuB was studied by determining protection of FhuB degradation by trypsin and proteinase K and by cross-linking experiments. His-tag-FhuD and His-tag-FhuD loaded with aerobactin specifically prevented degradation of FhuB and were cross-linked to FhuB. FhuD loaded with substrate and also FhuD free of substrate were able to interact with FhuB.     

The putative "switch 2" domain of the Ras-related GTPase, Rab1B, plays an essential role in the interaction with Rab escort protein

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the alpha2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative alpha2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the alpha3 and alpha4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B alpha2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B alpha2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the alpha2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.     

Intracellular calcium enhances the ethanol sensitivity of NMDA receptors through an interaction with the C0 domain of the NR1 subunit

Previous studies in this laboratory have shown that the ethanol inhibition of recombinant NMDA receptors expressed in Xenopus oocytes is subunit-dependent, with the NR1/2A receptor being more sensitive than NR1/2C receptors. The ethanol sensitivity of NR1/2A receptors is reduced by substitution of the wild-type NR1-1a (NR1(011)) subunit with the calcium-impermeable NR1 (N616R) subunit. In the present study, the ethanol inhibition of NMDA receptors was determined under different conditions to examine the role that calcium plays in determining the ethanol sensitivity of recombinant NMDA receptors. The ethanol sensitivity of NR1/2B or NR1/2C receptors was not affected by alterations in extracellular calcium levels or by coexpression with calcium-impermeable NR1 mutants. In contrast, the inhibition of NR1/2A receptors by 100 mM ethanol was reduced in divalent-free recording medium and was significantly increased when 10 mM calcium was used as the only charge carrier. The increase in the ethanol sensitivity of NR1/2A receptors under high-calcium conditions was prevented by preinjection of oocytes with the calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) but not by inhibitors of calmodulin or protein kinase C. Ethanol did not alter the channel blocking activity of divalent cations on NMDA-induced currents. The enhanced ethanol sensitivity of NR1/2A receptors in 10 mM calcium persisted when the NR1 subunit was replaced by the alternative splice variant NR1-4a (NR1(000)), which lacks the C1 and C2 cassettes. However, expression of a mutant NR1 subunit that lacked the C0, C1, and C2 domains abolished the calcium-dependent enhancement of ethanol's inhibition of NR1/2A receptors. Finally, the ethanol sensitivity of wild-type NR1/2A receptors measured in transfected HEK 293 cells by whole cell patch-clamp electrophysiology was significantly reduced by expression of the C-terminal truncated NR1 subunit. These results demonstrate that the ethanol sensitivity of certain NMDA receptors is modulated by an intracellular, calcium-dependent process that requires the C0 domain of the NR1 subunit.