Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.     

Channel properties of the purified glutamate receptor from rat brain reconstituted in planar lipid bilayer membrane

The properties of the purified rat brain glutamate receptor (GluR), reconstituted in planar lipid bilayer (BLM) were characterised. The single channel currents activated by glutamate and aspartate were similar. The different kinetics of current fluctuation were observed. Paroxysms of channel activity seems to be resulted from the transit of GluR through its active conformation from which it can open several times before desensitising. The effect of concanovaline A (Con A) as an agent blocking desensitisation of glutamatergic synapses was investigated. It was shown that Con A evokes high levels of conductivity and prolonged opening events of channels. Another agent, which stabilises glutamate activated conductivity, dithiothreitol (DTT), evokes "chronic" channel activity. This study demonstrates that purified GluR reconstituted in planar lipid bilayers exhibits the ion-conductivity properties that are associated with the postsynaptic membrane.     

Effects of carboxymethylation by a purified Torpedo californica methylase on the functional properties of the acetylcholine receptor in reconstituted membranes

The functional effects of carboxymethylation of Torpedo californica acetylcholine receptor by an endogenous Torpedo methylase were examined. Both the receptor and the methylase were purified to increase the level of methylation and the sensitivity of the functional assays. The methylase catalyzed the carboxymethylation of all four receptor subunits (alpha, beta, gamma, delta) with preferential labeling of the alpha and gamma subunits. For all the reactions, S-adenosylmethionine was used as the methyl donor. Functional effects of methylation were assessed by measuring ligand binding and ligand-activated ion permeability responses in reconstituted membranes containing purified acetylcholine receptors. Methylation of receptor to a level of 20 mol% had no significant effect on agonist or antagonist binding nor did methylation affect the transition from low-to-high affinity binding triggered by agonists. In contrast, 20% methylation led to a 20% reduction in the agonist-stimulated flux of cations across the receptor-containing membranes. The results suggest that methylation inhibits the ion permeability control properties of acetylcholine receptors.     

Affinity-labeling of purified acetylcholine receptor from Torpedo californica

The receptor for acetylcholine purified from electric tissue of $\text{Torpedo californica}$ has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.     

Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes

By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.     

Functional equivalence of monomeric and dimeric forms of purified acetylcholine receptors from Torpedo californica in reconstituted lipid vesicles

Acetylcholine receptors from Torpedo californica electric organ were solubilized and purified under conditions which prevent inactivation of the agonist-regulated cation channels. The dimer form of the receptors was preserved during purification. Treatment with reducing agents converted dimers into monomers. Receptor monomers and dimers were separately reconstituted into soybean lipid vesicles by the cholate dialysis technique. Reconstituted monomers and dimers were functionally equivalent with respect to their carbamylcholine-induced dose-dependent uptake of 22Na+, the total flux of 22Na+ per receptor during the permeability response, and the occurrence of desensitization. Evidence against non-covalent association of monomers to produce dimeric functional units was obtained using glutaraldehyde as a crosslinking agent. These results show that both the acetylcholine-binding sites and the agonist-regulated cation-specific channel are contained within the alpha 2 beta gamma delta subunit structure of the acetylcholine receptor monomer.     

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali- stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.     

Reconstitution of a partially purified endplate acetylcholine receptor preparation in lipid bilayer membranes

Incorporation of a fraction isolated from rat diaphragm muscle that contained the specific endplate cholinergic receptor into phospholipid bilayer membranes resulted in the production of an acetylcholine-stimulated conductance increment of large magnitude. The acetylcholine-stimulated conductance shows several characteristics of the $\text{in vivo}$ post-synaptic excitable system.     

Reconstitution of a partially purified endplate acetylcholine receptor preparation in lipid bilayer membranes

Incorporation of a fraction isolated from rat diaphragm muscle that contained the specific endplate cholinergic receptor into phospholipid bilayer membranes resulted in the production of an acetylcholine-stimulated conductance increment of large magnitude. The acetylcholine-stimulated conductance shows several characteristics of the $\text{in vivo}$ post-synaptic excitable system.     

Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.     

Effects of monoclonal antibodies on the function of acetylcholine receptors purified from Torpedo californica and reconstituted into vesicles

We tested the effects of 62 monoclonal antibodies (mAbs) to acetylcholine receptors from Torpedo californica on the function of receptor reconstituted into lipid vesicles. Two of these mAbs, mAbs 148 and 168, inhibited carbamylcholine-induced 22Na+ uptake into vesicles. The rate of 125I-labeled alpha-bungarotoxin (125I-alpha BGT) binding to the reconstituted liposomes was also reduced, although 125I-alpha BGT binding at equilibrium was not affected. Agonist-induced desensitization of the receptor was also affected by these mAbs. mAb 148 binds to the beta subunit of receptor, and mAb 168 binds to the gamma subunit. Both mAbs bind to the cytoplasmic surface of the receptor; correspondingly, both block function when added before reconstitution, and both were found to have no effect on function when added to preformed vesicles. Their effects were not due to interference with the reconstitution process. Both mAbs were capable of cross-linking receptors. In contrast to the bivalent mAbs, monovalent Fab fragments of these two mAbs had little effect on receptor function, which suggests that the effects of the bivalent mAbs resulted primarily from cross-linking receptors.     

Labeling of functionally sensitive sulfhydryl-containing domains of acetylcholine receptor from Torpedo californica membranes

N-(1-Pyrene)maleimide, a fluorescent, lipophilic, alkylating agent, was used as a probe for the nicotinic acetylcholine receptor (AChR). Preincubation with N-(1-pyrene)maleimide under nonreducing conditions inhibits agonist-induced cation permeability of AChR-enriched membranes. This inhibition is dependent on the concentration of N-(1-pyrene)maleimide used. This correlation was also exhibited by resonance energy transfer of tryptophan fluorescence to N-(1-pyrene)maleimide and by the labeling stoichiometries. However, agonist-induced desensitization, as based on the time-dependent inhibition of alpha-bungarotoxin binding upon preincubation with the agonist carbamylcholine, was unaffected by N-(1-pyrene)maleimide. Alkylation of the AChR by N-(1-pyrene)maleimide is pH-dependent with an apparent pKa of 7.5 and is unaffected by preincubation with carbamylcholine, alpha-bungarotoxin, tubocurarine, or decamethonium. Preincubation with a 25-fold molar excess of N-ethylmaleimide partially protects against N-(1-pyrene)maleimide, yet simultaneous incubation with an equimolar concentration does not protect. In contrast, simultaneous incubation with equimolar concentrations of phenylmaleimide or naphthylmaleimide inhibited N-(1-pyrene)maleimide alkylation by 52 and 67%, respectively. Each AChR subunit is labeled by N-(1-pyrene)maleimide. Prior alkylation with N-ethylmaleimide does not alter the labeling profile but lowers the amount of labeling of all subunits. Reductive methylation of membranes under conditions which dimethylate all or most protein amino groups does not inhibit alkylation by N-(1-pyrene)maleimide. The above results, as well as amino acid analysis of N-(1-pyrene)maleimide-alkylated receptor, indicate that a homologous class of cysteines, which reside in each subunit within the AChR domain embedded in the membrane, are involved in the reaction with N-(1-pyrene)maleimide.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

Monovalent ion effects on acetylcholine receptor from Torpedo californica

The effects of the five Group I monovalent ions, Li, Na, K, Rb, and Cs, on [³H]acetylcholine binding to Triton X-100 solubilized acetylcholine receptor from Torpedo californica electroplax were examined. Acetylcholine binding was not greatly affected by Li or Na, but was inhibited by the other ions in the order Cs > Rb > K. The inhibition by K appeared to occur by a mechanism identical to that for d-tubocurarine inhibition of acetylcholine binding.     

Disulfide bond cross-linked dimer in acetylcholine receptor from Torpedo californica

Acetylcholine receptor from $\text{Torpedo californica}$ electric tissue occurs in membrane, and is purified, as a mixture of monomer and dimer. Dimer is cross-linked by disulfide bonds involving one of the four polypeptide components of receptor, namely the one of apparent molecular weight of 64,000.     

Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers

An alpha-toxin-binding membrane protein, isolated from the head and thoracic ganglia of the locus (Locusta migratoria), was reconstituted into planar lipid bilayers. Cholinergic agonists such as acetylcholine, carbamylcholine, and suberyldicholine induced fluctuations of single channels, which suggests that the protein represents a functional cholinergic receptor channel. The antagonist d-tubocurarine blocked the activation of the channels, whereas hexamethonium had only a weak effect; similar properties have been described for nicotinic insect receptors in situ. The channel was selectively permeable to monovalent cations but was impermeable to anions. The conductance of the channel (75 pS in 100 mM NaCl) was independent of the type of agonist used to activate the receptor. Kinetic analysis of the channel gating revealed that, at high agonist concentrations (50 microM carbamylcholine), more than one closed state exists and that multiple gating events, bursting as well as fast flickering, appeared. At very high agonist concentrations (500 microM carbamylcholine), desensitization was observed. Channel kinetics were dependent on the transmembrane potential. Comparing the conductance, the kinetics, and the pharmacology of nicotinic acetylcholine receptor from insect ganglia and fish electroplax reconstituted into bilayers revealed obvious similarities but also significant differences.     

Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits.

Under conditions that limit proteolytic degradation, the detergent-solubilized purified receptor protein from Torpedo californica exists in monomeric and dimeric forms. The purified receptor complex is composed of four different polypeptide subunits of apparent molecular weights 40 000, 50 000, 60 000, and 65 000. The individual polypeptides have been purified and their amino acid compositions have shown them to be relatively hydrophobic. In addition, the carbohydrate composition of the intact receptor complex and of the individual polypeptides has been determined. Amino acid analysis provided evidence for the occurrence of a component with chromatographic properties similar to those of phosphoserine. Treatment of receptor with CH3NH2 in base, a condition which provided quantitative modification of O-phosphoserine residues in beta-casein, completely eliminated the peak corresponding to phosphoserine following mild acid hydrolysis. We conclude that the receptor contains O-phosphoserine residues to the extent of approximately seven residues per molecule and these residues occur in all constituent polypeptides. Other forms of O-substituted serine and threonine were also shown to occur, most likely as glycosylated residues.     

Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes.