A Possible Role for Ligatin and the Phosphoglycoproteins It Binds in Calcium-Dependent Retinal Cell Adhesion

Ligatin is a filamentous plasma membrane protein that serves as a baseplate for the attachment of peripheral glycoproteins to the external cell surface. Ligatin can be released from intact, embryonic chick neural retinal cells by treatment with 20 mM Ca⁺⁺ without adversely affecting their viability. α-Glucose-1-phos phate is also effective in removing ligatin-associated glycoproteins from intact cells. After either of these treatments, the retinal cells seem not to exhibit Ca⁺⁺ -dependent adhesion for one another. It is thus suggested that ligatin in neural retina may serve as a baseplate for the attachment to the cell surface of glycoproteins active in Ca⁺⁺-dependent adhesion. The finding that Ca⁺⁺ serves to protect Ca⁺⁺-dependent adhesion molecules from digestion by trypsin is discussed in relation to steric constraints on trypsin's accessibility to these adhesion molecules because of their possible binding to arrayed ligatin filaments.     

Immunochemical studies on blood groups. Structures and immunochemical properties of nine oligosaccharides from B-active and non-B-active blood group substances of horse gastric mucosae.

Oligosaccharides from base-borohydride-treated B-active and non-B-active glycoproteins of horse stomach mucosae were purified chromatographically on Bio-Gel P-2, charcoal-Celite, paper and high pressure liquid chromatography. From colorimetric and gas-liquid Chromatographic analyses, methylation, quantitative periodate oxidation and Smith degradation, structures of nine Oligosaccharides are proposed. Seven have not been previously described. The oligosaccharide isolated in largest amount in the B-active reduced tetrasaccharide analogous to an A-active reduced oligosaccharide from pig submaxillary mucin, and a reduced octasaccharide, the largest isolated, has two B determinants and may represent full expression of B-specific biosynthetic potential of the mucosal lining. Three B-active and one non-B-active oligosaccharide possessed the core structure previously identified in Oligosaccharides from human blood group, B, HLe^b, Le^a and precursor I substances. Two non-B-active and one B-active compound inhibited the cross reaction of type XIV horse antipneumococcal sera with blood group substances. Terminal nonreducing α-linked dGlcNAc (d-2-acetamido-2-deoxyglucopyranose), previously found in Oligosaccharides of hog blood group substances, was also present in a tetrassarcharide of the non-B-active material. Oligosaccharides released from blood group glycoproteins of horse stomach mucosae are smaller and hence less heterogeneous than those from human ovarian cyst and perhaps hog A + H and human gastric mucosae.     

Different sensitivities to agonist of muscarinic acetylcholine receptor subtypes

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

Control of the onset of migration of neural crest cells in avian embryos

Summary To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of Ca^{+ +} and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3–8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca^{+ +}- and Mg^{+ +}-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca^{+ +}-free medium duplicated the effects of CMF, but neither Mg^{+ +}-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane. Ca^{+ +}-channel antagonists D600 and Nifedipine, without any exernal Ca^{+ +}-depletion.The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca^{+ +}-depletion inactivates Ca^{+ +}-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca^{+ +}-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca^{+ +}-channels suggest that Ca^{+ +} levels may be involved in regulating this system.     

Identification of a gene family of cadherin cell adhesion molecules

Cadherins are a group of functionally related glycoproteins responsible for the Ca²⁺-dependent cell-cell adhesion mechanism. They are divided into subclasses, such as E-, P- and N-cadherin, which are distinct in immunological specificities and tissue distribution. Cell aggregation experiments suggest that these molecules have subclass specificities in cell-cell binding and are involved in selective cell adhesions. Analysis of amino acid sequences deduced from the nucleotide sequences of cDNAs encoding cadherins demonstrated that they are integral membrane proteins and share common sequences throughout their entire length; average similarity in the sequences among them is in a range of 50–60%. This result provided evidence that cadherins constitute a gene family which encodes adhesion molecules with different specificities. We also showed that, when cells with little cadherin activity were transfected with cadherin cDNAs, they acquired the cadherin-mediated adhesion properties.     

Calcium-dependent self-aggregation of toposome,a sea urchin embryo cell adhesion molecule

Summary— Sea urchin embryos can be easily dissociated into single cells by exposure to Ca²⁺- and Mg²⁺-free seawater. When transferred back to normal seawater, isolated cells spontaneously form aggregates capable of development. Here, the Ca²⁺-dependent self-aggregation of toposome, a 22S glycoprotein complex which mediates cell-cell adhesion in sea urchin embryos, has been investigated using the purified molecule. Results show that the 22S complex is completely converted to 15S particles by sedimentation on sucrose isokinetic gradients in the presence of EDTA. Reconstitution of the 22S complex is achieved by readdition of Ca²⁺. We propose that the 15S particle constitutes the toposome functional unit on the cell surface.     

Cadherin Expression in the Developing Vertebrate CNS: From Neuromeres to Brain Nuclei and Neural Circuits

Cadherins are a family of cell surface glycoproteins which mediate cell-cell adhesion by a Ca²⁺-dependent mechanism. Results from in vitro studies with cadherin-transfected cell lines show that cadherins preferentially bind to each other in a homophilic fashion. In the developing vertebrate brain, at least 10 cadherins are found. Some of these cadherins are expressed in a restricted fashion in particular developing brain nuclei and neural circuits. Based on these results, specific morphogenetic roles for cadherins during CNS development have been proposed. This review focuses on the possible role of cadherin-mediated sorting and aggregation of early neurons and neurites in the formation of brain nuclei, fiber tracts, and neural circuits. Moreover, at least 1 cadherin is also expressed in a segmental ("neuromeric") fashion in the early chicken forebrain, suggesting that this cadherin regulates developmental processes involved in the transformation from the neuromeric organization of the early neuroepithelium to the functional organization of the mature brain.     

Inhibitory effects of tunicamycin on cell-cell adhesion and cell reaggregation of the cellular slime mold,Polysphondylium pallidum

To assess the role in cell-cell adhesion of gp64, a putative cell-cell adhesion molecule ofPolysphondylium pallidum, we treated the cells with tunicamycin (TM), a known inhibitor of the synthesis of the N-linked oligosaccharide precursor, and examined TM's effect on cell-cell adhesion. The vegetative growth ofPolysphondylium cells was inhibited with TM in a dose-dependent manner. When cells were treated with TM (2.0 μg/ml) during only the first 4 hr of starvation and further starved for 8 hr without TM, the cells dissociated considerably, although even the growth phase cells ofPolysphondylium normally show EDTA-resistant (Ca²⁺-independent) cell adhesions. In parallel with the above effects, the amounts of intact gp64 decreased considerably in time with the lengths of incubation (0 hr>4 hr >8 hr). When TM-treated cells were washed free of TM, and shaken for a further 12 hr, the cells began to aggregate again, accompanied by an increase of gp64. In conclusion, TM affected cell-cell adhesion ofPolysphondylium cells, but we were not able to distinguish whether the inhibition of cell aggregation was due to defects in glycosylation on glycoproteins and/or due to reduced levels of glycoproteins themselves.     

Poorly Differentiated Colon Carcinoma Cell Lines Deficient in α-Catenin Expression Express High Levels of Surface E-cadherin but Lack Ca2+-Dependent Cell-Cell Adhesion

Studies on several different types of carcinomas, with the notable exception of colon carcinoma, have shown that poorly differentiated tumors are frequently deficient in E-cadherin dependent cell-cell adhesion. In this study, we examined Ca²⁺-dependent cell-cell adhesion in colon carcinoma cell lines. Five poorly differentiated (Clone A, MIP 101, RKO, CCL 222, CCL 228) and four moderately-well differentiated (CX-1, CCL 235, DLD-2, CCL 187) colon carcinoma cell lines were assayed for their ability to form cell-cell aggregates and for their levels of E-cadherin expression. All of the poorly differentiated cell lines exhibited low levels of Ca²⁺-dependent cell-cell aggregation, in contrast to the moderately-well differentiated cell lines. Contrary to most previous studies, however, we observed that three of the five poorly differentiated cell lines examined expressed E-cadherin by FACS analysis and immunoprecipitation using an E-cadherin mAb. In fact, two of these cell lines expressed a 3- to 4-fold higher level of E-cadherin than that found in the moderately-well differentiated cell lines. mRNA levels for E-cadherin, as evaluated by both RT-PCR and Northern hybridization, corresponded to the levels of protein expression in each of the cell lines. Immunoprecipitation with an E-cadherin mAb, which is known to co-precipitate the catenins, demonstrated that the three poorly differentiated cell lines expressing E-cadherin did not co-precipitate α-catenin, although all of the moderately-well differentiated cell lines expressed both α- and β-catenin. RT-PCR confirmed the absence of the α-catenin mRNA from two of these cell lines. Stable expression of an α-catenin cDNA in one of the poorly differentiated cell lines lacking α-catenin expression resulted in a 5-fold increase in its level of Ca²⁺-dependent cell-cell aggregation, providing evidence that α-catenin is directly responsible for the loss of cell-cell adhesion in some cell lines. The α-catenin transfectants also exhibited a marked reduction in migration on collagen I. These data indicate that loss of α-catenin expression, as well as E-cadherin expression, can lead to a phenotype associated with poorly differentiated colon carcinomas.     

Molecular biology of cadherins in the nervous system

Cadherins are cell-cell adhesion molecules belonging to the Ca²⁺-dependent cadherin superfamily. In the last few years the number of cadherins identified in the nervous system has increased considerably. Cadherins are integral membrane glycoproteins. They are structurally closely related and interspecies homologies are high. The function is mediated through a homophilic binding mechanism, and intracellular proteins, directly or indirectly connected to the cadherins and the cytoskeleton, are necessary for cadherin activity. Cadherins have been implicated in segregation and aggregation of tissues at early developmental stages and in growth and guidance of axons during nervous system development. These functions are modified by changes in type(s) and amount of cadherins expressed at different developmental stages. The regulatory elements guiding cadherin expression are currently being elucidated.     

Role of acetylcholine in Ca<Superscript>2+</Superscript>-dependent regulation of functional activity of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis> myocardium

To study role of acetylcholine (ACh) in Ca²⁺-dependent regulation of rhythm and strength of cardiac contractions in the frog Rana temporaria, we studied in parallel experiments the ACh chrono- and inotropic effects on the background of action of blockers of the potential-controlled Ca²⁺-channels, ryanodine and muscarine receptors. The obtained results indicate participation of acetylcholine in the Ca²⁺-dependent regulation of the rhythm and strength of the frog cardiac contractions.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Methionine Aminopeptidase II: A Molecular Chaperone for Sarcoplasmic Reticulum Calcium ATPase

The monoclonal antibody to the β-subunit of H⁺/K⁺-ATPase (mAbHKβ) cross-reacts with a protein that acts as a molecular chaperone for the structural maturation of sarcoplasmic reticulum (SR) Ca²⁺-ATPase. We partially purified a mAbHKβ-reactive 65-kDa protein from Xenopus ovary. After in-gel digestion and peptide sequencing, the 65-kDa protein was identified as methionine aminopeptidase II (MetAP2). The effects of MetAP2 on SR Ca²⁺-ATPase expression were examined by injecting the cRNA for MetAP2 into Xenopus oocytes. Immunoprecipitation and pulse-chase experiments showed that MetAP2 was transiently associated with the nascent SR Ca²⁺-ATPase. Synthesis of functional SR Ca²⁺-ATPase was facilitated by MetAP2 and prevented by injecting an antibody specific for MetAP2. These results suggest that MetAP2 acts as a molecular chaperone for SR Ca²⁺-ATPase synthesis.     

Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Cleavage stage mouse embryos share a common cell adhesion system with teratocarcinoma cells

We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca²⁺-dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca²⁺ but sensitive in the absence of Ca²⁺. When several embryos treated with trypsin in the presence of Ca²⁺ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca²⁺ but not without Ca²⁺. Embryos treated with trypsin in the absence of Ca²⁺ (TE) did not show such Ca²⁺-dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells.     

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-xβ in Xenopus oocytes

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca²⁺-dependent oscillatory Cl⁻ currents by acting through membrane-bound receptors. External application of 50 μM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca²⁺-dependent Cl⁻ currents were observed in the absence of extracellular Ca²⁺, were blocked by intracellular injection of the Ca²⁺ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca²⁺ ATPase. Intracellular Ca²⁺ release appeared to be from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store, because Cl⁻ currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein α subunits that were identified in Xenopus laevis; G_qα, G₁₁α, G₀α, and G_i1α. Among AS-ODNs against the Gαs tested, AS-G_qα and AS-G_i1α to S1P and AS-G_qα and AS-G₁₁α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca²⁺ from the IP₃-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998. © 1998 Wiley-Liss, Inc.     

Role of N-Cadherin cis and trans Interfaces in?the Dynamics of Adherens Junctions in Living Cells

Cadherins, Ca²⁺-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca²⁺-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca²⁺-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca²⁺-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.     

Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. V. Evidence that manganese inhibits a calcium-dependent step in programming for lysis

The mechanism of T-lymphocyte-mediated cytolysis consists of three successive steps: adhesion formation, programming for lysis, and killer-cell-independent lysis. Mg²⁺, but not Ca²⁺, is required for adhesion formation, whereas programming for lysis is strongly Ca²⁺ dependent. We have previously reported that the transition metal manganese can substitute for Mg²⁺ in supporting adhesion formation. In the present paper, we demonstrate that manganese inhibits programming for lysis. The inhibitory effect of Mn²⁺ on cytolysis can be reduced by increasing the concentration of Ca²⁺. Furthermore, inhibitor sequencing experiments were unable to distinguish the step blocked by Mn²⁺ from the Ca²⁺-dependent step. These results suggest that Mn²⁺ blocks a Ca²⁺-dependent step(s) in programming for lysis. Present evidence does not distinguish whether the action of Ca²⁺ in programming for lysis is via a Ca²⁺ influx (as a “second messenger?”) or whether Ca²⁺ simply serves as a cofactor at the cell exterior.     

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca²⁺ channel, in store-operated Ca²⁺ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3 and 5 rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-1 polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca²⁺ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP₃F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC₅₀ = 0.5 M), indicating that InsP₃F and LPA principally activate store-operated Ca²⁺ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP₃F-, LPA- or thapsigargin-stimulated Ca²⁺ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP₃F-stimulated Ca²⁺ inflow and only a very small enhancement of LPA-stimulated Ca²⁺ inflow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca²⁺ inflow through the previously-characterised Ca²⁺-specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.