A Calcium Channel from the Presynaptic Nerve Terminal of the Narke japonica Electric Organ Contains a Non-N-Type α2δ Subunit

Abstract: A monoclonal antibody (MCC-1) that recognizes the α₂δ subunit complex of L-type calcium channels from rabbit skeletal muscle membranes partially inhibited the evoked release of acetylcholine from synaptosomes isolated from the electric organ of the marine electric ray, Narke japonica . Digitonin extracts of synaptosomal plasma membranes were subjected to immunoaffinity column chromatography on MCC-1-Sepharose. The purified fraction contained a 170-kDa protein that reacts with MCC-1 and dissociates into smaller polypeptides under reducing conditions. In addition, immunoblotting analysis revealed the existence of syntaxin in the purified fraction, suggesting that the calcium channel forms a complex with syntaxin. However, MCC-1 did not immunoprecipitate an ω-conotoxin GVIA-binding protein. These findings indicate that the 170-kDa protein may be the α₂δ subunit of a calcium channel that is distinct from the ω-conotoxin GVIA-sensitive N-type calcium channel and partially responsible for the calcium influx that triggers the evoked release of acetylcholine.     

A novel α-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus

Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition-binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [(3) H]-GABA release is inhibited by α-conotoxin MII. However, the highly α6β2*-subtype-selective α-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-GABA release was unaffected by β3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxin MII-sensitive α3α4β2 nAChR. The remaining α-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs.     

Morphine enhances the phosphorylation of a 58 kDa protein in mouse brain membranes.

Morphine and [D-Ala2,D-Leu5]enkephalinamide enhance the phosphorylation of a 58 kDa protein in mouse brain synaptosomal membranes. The enhancement of phosphorylation was inhibited by naloxone, an antagonist of morphine. The phosphorylated 58 kDa protein was retained on wheat-germ-agglutinin-agarose and morphinone-Affi-Gel 401 columns and biospecifically eluted out from the columns with N-acetyl-D-glucosamine and naloxone respectively. These results suggest a strong possibility that the opiate-binding protein undergoes phosphorylation by endogenous protein kinase. Since the molecular mass of a mu-type opioid receptor in mouse brain is suggested to be 58 kDa, coincident with those of rat brain and neuroblastoma x glioma hybrid cells, it is conceivable that the phosphorylated 58 kDa protein is a mu-type receptor.     

Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse-tubule calcium channel

The tritiated arylazido phenylalkylamine (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4, 5-trimethoxyphenyl)-2-isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane-bound and purified calcium channel from guinea-pig skeletal muscle transverse-tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a Kd of 2.0 +/- 0.5 nM and a Bmax of 17.0 +/- 0.9 pmol/mg protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse-tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver-stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high-affinity drug receptor carrying subunit of the skeletal muscle transverse-tubule L-type calcium channel.     

A monoclonal antibody to the beta subunit of the skeletal muscle dihydropyridine receptor immunoprecipitates the brain omega-conotoxin GVIA receptor.

Antibodies against the subunits of the dihydropyridine-sensitive L-type calcium channel of skeletal muscle were tested for their ability to immunoprecipitate the high affinity (Kd = 0.13 nM) 125I-omega-conotoxin GVIA receptor from rabbit brain membranes. Monoclonal antibody VD2(1) against the beta subunit of the dihydropyridine receptor from skeletal muscle specifically immunoprecipitated up to 86% of the 125I-omega-conotoxin receptor solubilized from brain membranes whereas specific antibodies against the alpha 1, alpha 2, and gamma subunits did not precipitate the brain receptor. Purified skeletal muscle dihydropyridine receptor inhibited the immunoprecipitation of the brain omega-conotoxin receptor by monoclonal antibody VD2(1). The dihydropyridine receptor from rabbit brain membranes was also precipitated by monoclonal antibody VD2(1). However, neither the neuronal ryanodine receptor nor the sodium channel was precipitated by monoclonal antibody VD2(1). The omega-conotoxin receptor immunoprecipitated by monoclonal antibody VD2(1) showed high affinity 125I-omega-conotoxin binding, which was inhibited by unlabeled omega-contoxin and by CaCl2 but not by nitrendipine or by diltiazem. An antibody against the beta subunit of the skeletal muscle dihydropyridine receptor stained 58- and 78-kDa proteins on immunoblot of the omega-conotoxin receptor, partially purified through heparin-agarose chromatography and VD2(1)-Sepharose chromatography. These results suggest that the brain omega-conotoxin-sensitive calcium channel contains a component homologous to the beta subunit of the dihydropyridine-sensitive calcium channel of skeletal muscle and brain.     

Neural cell recognition molecule F11: membrane interaction by covalently attached phosphatidylinositol

The mode of membrane insertion of F11 130 kDa protein, a neural chick cell surface glycoprotein involved in neurite fasciculation, has been investigated. Up to 41% of total F11 130 kDa is released from adult chick brain plasma membranes by phosphatidylinositol specific phospholipase C (PI-PLC), whereas no release is mediated by lecithin/cephalin specific phospholipase C (PLC). PI-PLC dependent release of F11 is also observed from embryonal chick brain plasma membranes and from the surface of intact retinal cells. Biosynthetic labelling experiments demonstrate that F11 contains ethanolamine. Taken together, these results suggest that F11 interacts with the plasma membrane at least partially through covalently linked glycosyl-phosphatidylinositol (GPI) or a structurally similar lipid.     

Monoclonal Antibodies Recognizing the Benzodiazepine Receptor of Chick Embryo Brain

Abstract

The benzodiazepine receptor (BZDR) of the embryonic chick brain contained three subunit proteins with molecular weights of 48-kilodalton (KD), 50-KD and 51-KD at a pI of 5.6, as demonstrated by two-dimensional gel electrophoresis and fluorography of the ³H-flunitrazepam (FNZ)-photolabeled receptor. Monoclonal antibodies (mAB) against the receptor were produced by using the spleen cells of one mouse immunized with the three subunit proteins extracted from SDS-PAGE gels. When the radioligand-labeled membranes were subjected to two-dimensional gel electrophoresis followed by immunoblotting using the mAB 2C3, both 50-KD and 51-KD bands with a pI of 5.6 were immunoreactive and radioactive. Thus, the mAB 2C3 recognized a common epitope on the 50-KD and 51-KD subunits of the BZDR. In addition, the mAB 2C3 was used with immunocytochemistry to determine the distribution of the receptor in the chick embryo brain. The BZDR immunoreactivity was observed among various brain areas, including hippocampus, optic tectum and cerebellum. The reaction product was localized in the neuronal membranes and cytoplasm. Certain neurons in the culture derived from embryonic chick brains were also immunoreactive as detected by immunocytochemical staining.     

Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation.

Dihydropyridine receptors were purified from rabbit skeletal muscle transverse tubule membranes and incorporated into planar lipid bilayers. Calcium channels from both the purified dihydropyridine receptor preparation and the intact transverse tubule membranes exhibited two sizes of unitary currents, corresponding to conductances of 7 +/- 1 pS and 16 +/- 3 pS in 80 mM BaCl2. Both conductance levels were selective for divalent cations over monovalent cations and anions. Cadmium, an inorganic calcium channel blocker, reduced the single channel conductance of calcium channels from the purified preparation. The organic calcium channel antagonist nifedipine reduced the probability of a single channel being open with little effect on the single channel conductance. The presence of two conductance levels in both the intact transverse tubule membranes and the purified dihydropyridine receptor preparation suggests that the calcium channel may have multiple conductance levels or that multiple types of calcium channels with closely related structures are present in transverse tubule membranes.     

Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex

Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.     

Solubilization of the calcium antagonist receptor from rat brain

[3H]Nitrendipine binds with high affinity to a calcium antagonist receptor in rat brain membranes. At 4 degrees C, treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with [3H]nitrendipine. The nitrendipine concentration that gave a half-maximal amount of the solubilized [3H]nitrendipine-receptor complex was identical to the Kd for specific nitrendipine binding to brain membranes. Nitrendipine dissociated from digitonin-solubilized and membrane-bound receptors with a half-time of 24 to 30 min at 20 degrees C. Verapamil increased and diltiazem decreased the dissociation rate to a similar extent in both preparations indicating that the solubilized receptor contains both the dihydropyridine and diltiazem/verapamil binding sites. Sucrose gradient sedimentation experiments gave a value of S20, omega = 19.2 for the receptor-digitonin complex. The solubilized calcium antagonist receptor binds specifically to wheat germ agglutinin-Sepharose columns consistent with an identification as a glycoprotein.     

Bay K8644 like activity of an antibody against a 60 kDa tubular membrane protein

Partial purification of the dihydropyridine receptor from rat skeletal muscle demonstrated mainly a 60 kDa band in SDS-polyacrylamide gel. An antibody raised against that protein behaved as a calcium channel agonist viz. Bay K8644. The affinity purified antibody, when added to cultured heart cells, increased the beat rate 40-80% depending on the titer of the antiserum. The antibody also woke up the beats of the cells previously blocked with the channel antagonist, nifedipine. Immunoblot analysis indicated that the receptor of this antibody in heart cell membrane is also a 60 kDa protein.     

Serotonin Directly Increases a Calcium Current in Swim Motoneurons of Aplysia brasiliana

Muscle fibers in the swim appendages of the mollusk Aplysiabrasiliana are innervated by cholinergic motoneurons. Serotonin(5-HT) causes an increase in amplitude of junctional potentialsand muscle contractions at this neuromuscular synapse. We studiedmotoneurons with intracellular current-clamp recording and single-electrodevoltage-clamp analysis to determine the effects of 5-HT on somaticcurrents in these presynaptic neurons. Serotonin was found tohave no effect on action potential duration in motoneurons bathedin normal seawater, and no effect of 5-HT could be detectedon K⁺ currents, indicating that 5-HT does not indirectly enhancecalcium currents by prolonging the action potential. Calciumcurrents were isolated by replacing extracellular sodium withTEA and adding tetrodotoxin and other K⁺-channel blockers. Underthese conditions motoneuron action potentials were greatly prolongedand could be blocked with Co²⁺ or Cd²⁺. Addition of 5-HT increasedthe duration of these Ca²⁺ spikes by about 35%. In motoneuronsstudied with voltage clamp, the amine produced a 58% increasein total inward calcium current. Use of the calcium channelblockers nifedipine, nimodipine, -conotoxin GVIA, and -agatoxinTK revealed that motoneurons express varying amounts of L-,N- and P-like calcium channels, but only an agatoxin-sensitive,P-type channel is sensitive to 5-HT. It is concluded that 5-HTacts directly to increase a P-type Ca²⁺ current during a normalspike. The resulting increase in intracellular calcium couldcontribute to an increase in transmitter release and accountfor the increase in junctional potentials in swim muscles.     

Proportions of Ca2+ Channel Subtypes in Chick or Rat P2 Fraction and NG108-15 Cells Using Various Ca2+ Blockers

The proportions of calcium (Ca²⁺) channel subtypes in chick or rat P₂ fraction and NG 108-15 cells were investigated using selective L-, N-, P- and P/Q- type Ca²⁺ channel blockers. KCl-stimulated ⁴⁵Ca²⁺ uptake by chick P₂ fraction was blocked by 40~50% using N-type Ca²⁺ channel blockers [-conotoxin GVIA, aminoglycoside antibiotics and dynorphin A(1–13)], but was not inhibited by P- or P/Q-type blockers (-agatoxin IVA or -conotoxin MVIIC). On the other hand, KCl-stimulated ⁴⁵Ca²⁺ uptake by rat P₂ fraction was blocked by 30~40% using P- or P/Q-type Ca²⁺ channel blockers, but was not inhibited by N-type Ca²⁺ channel blockers. The L-type Ca²⁺ channel blockers 1,4-dihydropyridines, diltiazem and verapamil, but not calciseptine (CaS), inhibited both KCl-stimulated ⁴⁵Ca²⁺ uptake and veratridine-induced ²²Na⁺ uptake by chick or rat P₂ fraction with similar IC₅₀ values. CaS did not have any effect on ⁴⁵Ca²⁺ uptake by either chick or rat P₂ fraction. In NG108-15 cells, CaS, -agatoxin IVA and -conotoxin MVIIC, but not -conotoxin GVIA, inhibited KCl-stimulated ⁴⁵Ca²⁺ uptake by 30–40%. Various combinations of these Ca²⁺ channel blockers had no significant additional effects in chick or rat P₂ fraction or NG 108-15 cells. These findings suggest that KCl-stimulated ⁴⁵Ca²⁺ uptake by chick or rat P₂ fraction and NG 108-15 cells is a convenient and useful model for screening whether or not natural or synthetic substances have selective effects as L-, N-, P-, or P/Q- type Ca²⁺ channel antagonists or agonists.     

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.     

Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.     

8-Bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes

Superfusion with 8-bromo-cyclic GMP or intracellular injection of cyclic GMP inhibits calcium-dependent slow action potentials in embryonic chick or guinea pig ventricular cells, suggesting that cyclic GMP inhibits calcium currents. Recently, cyclic GMP has been shown to reduce cyclic AMP-stimulated calcium currents in voltage-clamped ventricular myocytes. Since earlier results in intact cells had suggested that cyclic GMP might inhibit basal (i.e., unstimulated by cyclic AMP) calcium currents, we directly investigated the effect of 8-bromo-cyclic GMP on basal calcium channel currents (using barium as the charge carrier) in voltage-clamped ventricular myocytes isolated from embryonic chick hearts. Superfusion with 1 mM 8-bromo-cyclic GMP (without prior cyclic AMP elevation) progressively decreased peak calcium channel currents (-68% at 15 min after the onset of drug exposure). In contrast, the currents were unchanged during 15 min superfusion with control solution, or 1 mM 8-bromo-GMP (the noncyclic inactive analog of 8-bromo-cyclic GMP). The present results in voltage-clamped embryonic chick heart cells indicate that cyclic GMP can inhibit basal calcium channel currents, apparently through a cyclic AMP-independent mechanism.     

Blockade of ion channel expression in Xenopus oocytes with complementary DNA probes to Na+ and K+ channel mRNAs

Ionic currents were recorded from Xenopus oocytes injected with RNA isolated from chick or mouse brain. Three currents were studied: a rapid tetrodotoxin-sensitive Na+ current (Ina), an early outward K+ current sensitive to 4-aminopyridine (IA), and an inward current activated by the excitatory amino acid receptor agonist kainate. Oligonucleotides (60-80 bases long) complementary to rat brain Na+ channel sequences were prehybridized to chick brain RNA. These DNA sequences, upon injection into oocytes, specifically inhibited expression of INa relative to IA and the kainate-induced current in a dose-dependent manner. By contrast, prehybridization of oligonucleotides complementary to sequences either from the Drosophila Shaker locus (which codes for an early K+ current in Drosophila muscle) or from a homologous clone from mouse brain did not block the expression of the early outward K+ current induced in the oocytes by mRNA from chick or mouse brain. This method provides a convenient means for testing the functional role of cloned DNA species.     

Monoclonal antibodies immunoprecipitating omega-conotoxin-sensitive calcium channel molecules recognize two novel proteins localized in the nervous system.

Two monoclonal antibodies raised against brain synaptic membranes immunoprecipitated significant fractions of the brain omega-conotoxin receptor (probable omega-conotoxin-sensitive calcium channels) solubilized with digitonin. These antibodies recognized different proteins of 36 kDa and 28 kDa, respectively. Immunoblot analysis of fractions obtained by sucrose gradient centrifugation suggested that these two proteins were not subunits of the omega-conotoxin receptor but were bound to it. These proteins were found to be conserved at least from an amphibian to mammals, and to be present in the nervous system and adrenal medulla among the tissues examined.     

Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier

The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.     

Isolation and partial characterization of high affinity laminin receptors in neural cells

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.