Characterization of the Nicotinic Acetylcholine Receptor Isolated from Goldfish Brain*

We have studied the binding of a-bungarotoxin to a particulate fraction of goldfish brain enriched in syn- aptosomes. The binding is specific and saturable and exhibits the pharmacological properties of a nicotinic cholinergic receptor. Equilibrium binding measure- ments yield a single dissociation constant (KD) of 0.92 XIM. Kinetic analysis revealed one association rate con- stant and two dissociation rate constants. Dissociation constants calculated from kinetic measurements were 1.9 IIM and 12.5 PM. The toxin*receptor complex is readily solubilized in nonionic detergent. The isoelectric point of the toxin=receptor complex was found to be 5.00 + 0.01. Sedimentation velocity analysis in su- crose/HzO and sucrose/DzO gradients in conjunction with Sepharose 4B chromatography and diffusion experiments yielded a sedimentation constant of 11.45, a partial specific volume of 0.79 cm3/g for the toxin* receptor l detergent complex, and a molecular weight of approximately 340,000 for the toxin*receptor complex.

The elapid neurotoxin, u-bungarotoxin has been shown to bind specifically and saturably to membrane fragments from muscle cells (1, 2), electroplax (3), sympathetic ganglia (4), and both vertebrate (5-8) and invertebrate (9) brain. The fact that a-Btx' inhibits the agonist-induced activation of acetylcholine receptors of muscle and electroplax (10) is well known. Several reports have suggested that the a-Btx binding component of sympathetic ganglia (11,12) and cultured sympathetic neurons (13-15) may not be an acetylcholine receptor because of the inability of a-Btx to block agonist-induced physiological responses. Recent studies from our laboratory, however, suggest that the optic tecta of both the marine toad, Bufo marinus (16,17), and the common goldfish, Carassius auratus (la),' con- tain acetylcholine receptors that are sensitive to a-Btx. In the toad, the intracellularly recorded response of tectal cells to iontophoretically applied acetylcholine and to the native optic nerve transmitter is abolished by a-Btx." In addition, the postsynaptic portion of the visually evoked tectal "off" response, identified by the technique of current source-density analysis (19), is abolished by the topical application of a-Btx. Similarly, in the goldfish, the postsynaptic responses of tectal neurons elicited by all three classes of optic nerve fibers are abolished by the micropipette injection of n-Btx into the tectum (18)." The goal of the present experiments was to characterize the ru-Btx binding protein of goldfish brain. Knowledge of the binding parameters and molecular properties of the binding protein are of interest because the molecule is very likely the acetylcholine receptor. Also, Freeman (17) has provided evidence for the role of the acetylcholine receptor in the maintenance of synaptic connections. Thus, the binding protein is of interest not only because of its involvement in ion translocation but also because of its possible role in neuroplasticity.
In this paper, we present evidence that the cu-Btx binding protein of goldfish brain has kinetic properties different from those of the n-Btx binding protein of sympathetic ganglia (4), but quite similar to those of the acetylcholine receptors from muscle (1) and electroplax (20). We also present data on the pharmacological specificity, the isoelectric point, and molecular. size of the binding protein.
where Ro is the initial receptor concentration and TO is the initial toxin concentration.
When kl was calculated at nonsaturating levels of toxin, Ro was calculated from the following expression: To -RT,

Toxin-Receptor
Interaction-The amount of '""I-cr-Btx bound to particulate fractions of goldfish brain is a saturable function of the amount of toxin added. Fig. 2 shows the results of a typical experiment.
The K/j calculated from experiments using 5 mg to 25 mg wet weight equivalent per assay tube was 0.917 + 0.020 nM. In contrast to the report of Speth et al. (31), no dependence of KI, on the amount of tissue present was observed.
The dissociation of toxin. receptor complexes was studied by the addition of a 1000-fold excess of cold cu-Btx after a saturating dose of ""I-cY-Btx had equilibrated with the receptor. Aliquots were removed at various times following the addition of cold e-Btx and the amount of bound ""I-o-Btx was measured. To control for the effects of proteolysis and denaturation a parallel set of measurements in which buffer replaced cold a-Btx was made. The results, shown in Fig. 3, are expressed as normalized binding, i.e. binding observed when excess cold cu-Btx was added following equilibration divided by binding when no cold cu-Btx was added. The semilog plot of Fig. 3 demonstrates clearly that two rates of dissociation are present. The fast dissociation site has a first order dissociation constant of 6.57 X 1O-4 s-', yielding a half-time of 17.6 min. It comprises approximately 20% of the toxin.receptor complexes. The slow dissociation site has a dissociation con-  ""I-e-Btx was bound to particulate fractions, extracted with 1% Emulphogene BC-720, and loaded into a prefocused pH 4 to 6 gradient. The peak eluting with the anode solution is free ""I-a-Btx. The isoelectric point of the toxin. receptor complex is 5.00 f 0.01. stant of 3.456 + 0.391 X lo" M-' s-'. Using this association rate constant with the dissociation rate constants calculated above, two equilibrium dissociation constants can be calculated. The "low affinity site" has a KI, of 1.90 nM and the "high affinity site" has a Ku of 12.47 PM. The equilibrium binding is dominated by the "low affinity site" because the assay is not sufficiently sensitive to determine accurately an equilibrium dissociation constant of that low magnitude.
The equilibrium dissociation constant in the range of toxin concentrations studied should, however, be a composite of the dissociation constants for both sites. It can be shown that:" K,, = kKti + KI. k+l which relates the Kn obtained from Scatchard analysis or direct fit to RT = Roe T/(T + K,,) to the two known dissociation constants obtained from kinetic analysis. KH refers to the dissociation constant for the high affinity site, K,. to the low affinity constant, and /z to the ratio of the number of high to low affinity sites. Using this expression with the dissociation constants calculated above from the kinetic experiments yields a K,, of 0. 39 4 presents the data using the log-log plot described under "Experimental Procedures." The data for decamethonium are not shown because they had no effect on the equilibrium binding of '""I-a-Btx at concentrations lower than 10 mM. Table I summarizes the K, values determined from the log Z axis and the number of ligand binding sites per toxin binding site calculated from the slope of the line.
Isoelectric Focusing-The n-Btx binding protein of goldfish, like that of Torpedo (33) and muscle (34), is an acidic molecule.
Six preparative isoelectric focusing experiments yielded a value for the isoelectric point of 5.00 +-0.01. In each of the six experiments, the toxin *receptor complex was added to a different portion of either a pH 4 to 6 or a pH 3 to 10 ampholine gradient. Fig. 5 shows the results of an experiment using a pH 4 to 6 gradient.
Molecular Characterization-Chromatography of the toxin. receptor complex on Sepharose 4B is shown in Fig. 6  where Zz is the Boltzmann constant, n is the viscosity of the medium, and T is the absolute temperature. The value of D20.u. for the toxin. receptor complex from gel filtration is 2.51 X lo-' cm'/s. The diffusion coefficient of the toxin. receptor. detergent complex was also measured using the free diffusion technique (25). A typical experiment is illustrated in Fig. 7. D20.rc. was calculated from the observed diffusion coefficient of the complex relative to a series of standard proteins (Fig. 8). The D2,,,,, for the complex was 2.851 f 0.541 x lo-' cm'/s. When the standards were plotted in terms of Stokes radius, the Stokes radius of the toxin.receptor complex is 74.3 + 4.3 A (95% confidence intervals).
The sedimentation profile in sucrose/Hz0 of the toxin. receptor complex is shown in Fig. 9. As shown, the major component running near catalase on the gradient specifically binds ""I-cY-Btx, as demonstrated by the fact that it is not 'Z"I-u-Btx was bound to the receptor before solubilization.
The amount of complex in each tube was determined by an ammonium sulfate precipitation described under "Experimental Procedures." The diffusion coefficient was determined with nonlinear least squares using Simpson's rule for numerical integration. present when the tissue homogenate is preincubated with unlabeled cu-Btx. The sedimentation analysis described under "Experimental Procedures" is shown in Fig. 10. The curves for sucrose/HgO and sucrose/D20 gradients intersect at a value of spg,, of 11.45 and $* of 0.786 cm,'/g. The sedimentation data, in conjunction with the Stokes radius calculated from either the Sepharose 4B column or the diffusion analysis, allow the calculation of the molecular weights of both the protein-detergent complex and the protein portion of the complex. The frictional coefficient (f/f;,) of the protein. detergent complex can also be determined.
These data are summarized in Table II

DISCUSSION
The cu-Btx binding protein of goldfish brain exhibits many properties of the acetylcholine receptor from muscle (1) and electroplax (3). The cu-Btx binding protein of sympathetic ganglia (4) can be kinetically distinguished from that of electroplax, muscle, and goldfish brain. Equilibrium binding measurements, however, are similar. For goldfish, toxin-receptor interactions yield a Ku of 0.92 X lo-" M, revealing only a single class of toxin binding sites with Scatchard (not shown) and double reciprocal (Fig. 2) analysis. A similar value for the dissociation constant of 1.1 x IO-" M was observed by Greene (4) in sympathetic ganglia. Association rate constants for the binding of a-Btx are quite similar for Torpedo californica electroplax (1.9 X IO" Mm' s-l, (40)), rat muscle (1.1 to 1.5 X lo" M-' s-', (l)), and goldfish brain (our value of 3.5 X 10' Mm' s-') but are somewhat slower for sympathetic ganglia (4.3 X lo4 Me' s-' (4)). An additional, very rapid association component was observed for both muscle (4 to 6 X 10" Mm' se', (1)) and electroplax (1.3 X 10' Mm' SC', (40)). An important distinction between the cu-Btx binding component of sympathetic ganglia, which does not seem to be involved in chemical synaptic transmission, and the n-Btx binding components of electroplax, muscle, and goldfish brain, which are involved in synaptic transmission, is the dissociation kinetics of a-Btx. Both Patrick and Stallcup (14), using a rat sympathetic nerve line, and Greene (4), using chick sympathetic ganglia, have observed a single dissociation rate of 4.9 x 10m5 s-' and 4.6 X 10e5 s-', respectively. In goldfish, muscle, and electroplax, on the other hand, dissociation of the complex is much slower. In both goldfish brain and rat muscle, cu-Btx exhibits two distinct dissociation rate constants. The fast component has a rate constant of 4.8 X 10m5 for rat muscle (1) and 6.6 x 10e4 s-' for goldfish brain. The slow component has a dissociation constant of 1.9 x lOWe for rat muscle (1) and 4.3 x lo-" s-' for goldfish brain. The dissociation of a-Btx from electroplax is too slow to be measured (3,40). Maelicke et al. (20) have thoroughly characterized the kinetic and equilibrium interactions between the cu-neurotoxin of Naja Naja siamensis venom and the acetylcholine receptor from Electrophorus electricus electroplax. The a-neurotoxin of Naja Naja siamensis is homologous with a-Btx (41) and has similar biological properties.
In a number of elegant experiments, Maelicke et al. (20) found that the two dissociation rates arise from the existence of paired toxin binding sites which are interconvertable between high and low affinity states. Our data do not distinguish between the presence of paired interconvertable sites and the presence of two distinct a-Btx binding molecules with similar association kinetics but different dissociation kinetics; however, studies are presently underway to do so.
In systems such as electroplax, muscle, and toad and goldfish brain, where a-Btx inhibits the activation of the AChR by nicotinic agonists, the dissociation kinetic data include a component with a half-time of dissociation of greater than 40 h; whereas, sympa 'letic ganglia which bind a(-Btx but whose AChR activation is insensitive to it exhibits simple dissociation kinetics with a half-time on the order of 4 h. This suggests that measurement of the dissociation kinetics of the o-Btx. nAChR complex might provide a useful estimate of its physiological effectiveness in blocking ACh-induced ion flux at other synapses. The pharmacological properties of the goldfish brain a-Btx   (14), and electroplax (20). The inhibition of a-neurotoxin binding by hexamethonium in goldfish brain was similar to that in electroplax (20) but was much more potent than in sympathetic ganglia (43) and mammalian brain (31). The failure of decamethonium to inhibit binding, on the other hand, was more similar to rat brain and sympathetic ganglia than to electroplax. Isoelectric focusing of the toxin. receptor complex revealed that like the electroplax (33) and rat muscle (34) nAChR, the goldfish a-Btx binding protein is an acidic molecule with an isoelectric point of approximately 5.0 for the nAChR-cw-Btx complex.
Studies are currently under way to determine whether denervation by severing the optic nerve might produce a de nouo synthesis of receptor species in the optic tectum of the goldfish with a different isoelectric point, as is observed in muscle following denervation (34). The method of Smigel and Fleischer (23) used in the present study to characterize the molecular parameters of the goldfish brain a-Btx binding protein involves a number of assumptions. The calculation of the Stokes radii requires that the standards that are used do not bind appreciable detergent, in this case Triton X-100. This assumption appears reasonable from the work of Helenius and Simons (44) showing that hydrophilic proteins bind little or no Triton X-100. In addition, the method assumes that the mole fraction of bound detergent (Xl]) is independent of sucrose concentration and of the use of HZ0 uersus D20 as the solvent. The value of 0.735 cm"/g for a typical, nonglycosylated protein is assumed. This assumption must be considered tentative because at the present time, we have no information concerning whether or not the goldfish cw-Btx binding protein is glycosylated. The assumption is made that all of the membrane lipids were replaced by detergent. The validity of this assumption cannot be determined without a purified protein sample.  Meunier et al. (45) for Electrophorus AChR (12.5 S) and larger than that reported by Devreotes et al. (46) for chick muscle nAChR of 10.5 S. The Stokes radius was determined using both gel filtration and diffusion analysis because of the discrepancies observed with asymmetric particles (47). The two techniques yielded a value of approximately 80 8, for the toxin .receptor. detergent complex. Assuming a value OF 0.735 cm3/g for the U of the protein portion of the molecule, approximately 70% of the complex is found to be protein. This yields an average molecular weight for the toxin.receptor complex of 340,000. The frictional coefficients suggest that the molecule behaves as either a prolate ellipsoid with an axial ratio between 5.5 and 8 or an oblate ellipsoid with an axial ratio between 7.5 and 10.5. The value of the frictional coefficient was obtained using the assumption that the degree of hydration (6) was 0.2 g of solvent/g of protein as suggested by Tanford (35). The accuracy of the axial ratio estimates is thus dependent on the validity of this assumption.
The biochemical characterization of the goldfish brain (Y-Btx binding protein presented in this paper in conjunction with physiological studies demonstrating the ability of a-Btx to block synaptic transmission in the goldfish optic tectum provides compelling evidence that a-Btx binds to a nicotinic cholinergic receptor protein. Using the regenerating goldfish retinotectal system, studies are currently under way to determine what role the a-Btx protein might play in the formation and maintenance of synaptic connections and to determine any dynamic changes in the protein as a function of denervation and reinnervation.