Sodium transport by the acetylcholine receptor of cultured muscle cells.

Activation of the acetylcholine receptors of cultured muscle cells by carbamylcholine increases the rate of passive 22-Na+ uptake into the muscle cells up to 20-fold. The Na+ transport activity of the receptor desensitizes during exposure to carbamylcholine. The rate and extent of desensitization is reduced by lowering the assay temperature from 36 degrees to 2 degrees, allowing accurate measurements of initial rates of Na+ transport by the receptor. Activation of the receptor by carbamylcholine and acetylcholine is significantly cooperative (Hill coefficients of 1.4 to 2.0). Inhibition by D-tubocurarine is not cooperative. The carbamylcholine-induced Na+ transport activity of the receptor is inhibited 50% by 4 muM D-tubocurarine, 100 muM atropine, or 1.6 nM diiodo-alpha-bungarotoxin but is not affected by tetrodotoxin. The initial rate of Na+ transport by the receptor is temperature-independent between 2 degrees and 36 degrees. Receptor Na+ transport is saturable by Na+ at 2 degrees with an apparent Km of 150 plus and minus 20 mM. Saturation by Na+ not observed at 36 degrees at the concentrations tested. Saturation by Na+ is observed at 2 degrees both under conditions of net Na+ influx and under conditions of isotopic exchange at equilibrium. The receptor does not catalyze obligatory exchange diffusion at a detectable rate. Comparison of binding of [125-I]diiodo-alpha-bungarotoxin with rates of Na+ transport indicates a turnover number of 2 times 10-7 ions per min per receptor. These results are discussed in terms of the mechanism of Na+ transport by the receptor.


Activation
of the acetylcholine receptors of cultured muscle cells by carbamylcholine increases the rate of passive zzNa+ uptake into the muscle cells up to ZO-fold.
The Na+ transport activity of the receptor desensitizes during exposure to carbamylcholine.
The rate and extent of desensitization is reduced by lowering the assay temperature from 36" to 2", allowing accurate measurements of initial rates of Na+ transport by the receptor. Activation of the receptor by carbamylcholine and acetylcholine is significantly cooperative (Hill coefficients of 1.4 to 2.0). Inhibition by n-tubocurarine is not cooperative. The carbamylcholine-induced Na+ transport activity of the receptor is inhibited 50% by 4 PM n-tubocurarine, 100 PM atropine, or 1.6 nM diiodo-cy-bungarotoxin but is not affected by tetrodotoxin.
The initial rate of Na+ transport by the receptor is temperature-independent between 2" and 36". Receptor Na+ transport is saturable by Na+ at 2" with an apparent K, of 150 =t 20 mm Saturation by Na+ is not observed at 36" at the concentrations tested. Saturation by Na+ is observed at 2" both under conditions of net Naf influx and under conditions of isotopic exchange at equilibrium.
The receptor does not catalyze obligatory exchange diffusion at a detectable rate. Comparison of binding of [1251]diiodo-cy-bungarotoxin with rates of Naf transport indicates a turnover number of 2 X 10' ions per min per receptor.
These results are discussed in terms of the mechanism of Na+ transport by the receptor.
During neuromuscular transmission of impulses, acetylcholine released from the presynaptic nerve ending binds to acetylcholine receptors of nicotinic specificity located in the postsynaptic muscle membrane and causes an increase in the passive permeability of the postsynaptic muscle membrane to Na+, Kf, and other small cations.
Electrophysiological studies have described both the presynaptic and postsynaptic events by monitoring transmembrane potentials and currents with microelectrodes placed within the cells (for a review see Reference 1). The postsynaptic response to acetylcholine is inhibited specifically by * Recipient of Research Fellowship from the Muscular Dystrophy Association of America. the (Y toxins of elapid snakes (Bungurus, Naja nczja) and these toxins have been used to demonstrate the presence in the postsynaptic membrane of a specific class of macromolecules responsible for the response to acetylcholine (2).
Myoblasts obtained from embryos of a number of species can be cultured in z&o.
During growth in vitro the cells divide and fuse into multinucleated myotubes (3) which are crossstriated, contain hypolemmal nuclei, contract spontaneously, and are capable of synapse formation (4, 5). Myotubes formed in vitro have a high concentration of acetylcholine receptors measured either electrophysiologically (5-7) or by binding of snake cy toxins (8,9). Cultured muscle cells therefore provide an important model system for studies of receptor function and regulation. Although microelectrode techniques involving recording from single cultured cells have yielded much useful information, convenient ion transport methods for studying directly the permeability changes due to receptor activation would be useful. Kasai and Changeux (10) have reported extensive, studies of 22Naf efflux catalyzed by acetylcholine receptors in membrane vesicles from eel electroplax and demonstrated good pharmacological correlation between isotope flux measurements on membrane vesicles and electrophysiological current measurements on intact electroplax.
In the present investigation, I have applied a modification of the ion transport approach of Kasai and Changeux to a study of the Na+ permeability increase due to activation of the acetylcholine receptors of cultured muscle cells. Effect of ouabain and carbamylcholine on 2*Na+ uptake. *2Na+ uptake was measured at 36" as described under "Experimental Procedure" in assay medium ( l ), assay medium plus 5 mM ouabain (O), or assay medium plus 5 mM ouabain and 1 mM carbamylcholine 5% horse serum, 2yo fetal bovine serum, 2% chick embryo extract, 50 units/ml of penicillin, and 10 pg/ml of streptomycin at 36.5' in an atmosphere of 10% C02/90% air. Cultures were used after 5 to 9 days in vitro with medium changes every 2 to 3 days. In some experiments cultures were treated for 48 hours with 10 pM n-arabinofuranosylcytosine on day 3 or 4 to reduce the fibroblast population (4). Results obtained with such cultures did not differ qualitatively from those with untreated cultures. The growth medium of all cultures was supplemented with 0.2 &X/ml of [3H]leucine 48 hours prior to use in experiments to allow use of 3H counts per min as a measure of protein recovery in transport experiments.
Uptake was initiated by addition of assay medium containing **NaCl (5 to 20 pCi/ml) and the effecters noted in the figure legends. Uptake was terminated by removing the radioactive uptake medium and washing three-times at 20 with nonradioactive wash medium consistins of 164 mM NaCl. 5.4 mM KCl. 1.8 mM CaC12, 0.8 mM MgSO+ 5.0 rni N-2-hydroxyethylpiperazine: N'-2-ethanesulfonic acid (adjusted to pH 7.4 with NaOH), and 1 mM n-tubocurarine to inhibit receptor-catalyzed ion movements. Cell monolayers were then suspended in 0.4 N NaOH and radioactivity was measured in a scintillation counter. Uptake rates in nanomoles/min/mg of cell protein were calculated from the measurements of 2*Naf taken up and determinations of cell protein by a modification of the method of Lowry et al. (14). Values were corrected for variable protein recovery during the assay and washing procedures on the basis of the [3H]leucine radioactivity recovered in each sample.
Binding experiments were carried out in assay medium supplemented with 2 mg/ml of bovine serum albumin.
Excess toxin was washed off with five 1-min washes of the same medium. cells were suspended in 0.4 N NaOH, and radioactivity measured in a well counter.
In all experiments, toxin binding was inhibited more than 90% by 200 PM n-tubocurarine.

Measurement
of Sodium Permeability Increase Caused by Carbamylcholine-Measurements of the passive Na+ permeability of cultured muscle cells have been carried out essentially as described previously (13) by inhibiting the active extrusion of Na+ catalyzed by sodium and potassium-activated ATPase ((Na+ + I(+)-ATPase) with ouabain.
In the absence of ouabain, the cultured muscle cells take up only small quantities of nNa+ in exchange with the small pool of nonradioactive Na+ within the cells (Fig. 1). In the presence of ouabain, much larger quantities of 22Na+ are taken up (Fig. 1)  2. Time course of receptor Na+ transport activity. 22Na+ untake was measured at 36" as described under "Exnerimental Procedure" in the presence of 5 mM ouabain (0) or 5 I& ouabain and 1 mM carbamylcholine (0). The data are plotted on semilogarithmic coordinates (13).
within the cells increases to the medium concentration (152 mM) . The half-time for passive equilibration is 20 to 30 min and the time course is approximately exponential.
Comparison of the extent of nNa+ uptake in the presence and absence of ouabain suggests that the cells maintain an internal Na+ concentration lkfold lower than the medium concentration or approximately 13 mM. Carbamylcholine (1 mM) causes a substantial increase in the initial rate of 22Na+ uptake (Fig. 1). This increase is completely inhibited by 100 pM n-tubocurarine or 6 nM ar-bungarotoxin (see below) and therefore must reflect activation of the nicotinic acetylcholine receptors of the cultured muscle cells. The increase in initial rate at early time points (Fig. 1, inset) is l&fold in this experiment.
Although the increase in 22Na+ uptake is very large at early times, the initial rate of uptake is not maintained.
This point is illustrated more clearly in Fig. 2, in which some of the data of Fig. 1 have been plotted on semilogarithmic coordinates. If the permeability of the cells to Na+ is constant throughout the equilibration, the time course should be logarithmic and a plot of In ([22Na+]z/([22Na+]yn -["Na+&)) versus time should give a straight line whose slope is lr~Z/rr/~ (13). This relation is fulfilled in the absence of carbamylcholine (Fig. 2). In the presence of carbamylcholine, however, the rate of uptake is initially rapid (dashed line, Fig. 2) but decays to the control rate within 2 min. Thus the Na+ transport activity of the acetylcholine receptors of cultured muscle cells desensitizes during continued exposure to carbamylcholine or acetylcholinel as has been described for receptors at adult neuromuscular junction (15).
The rate of desensitization of the Na+ transport activity of the receptor can be measured by preincubation with carbamylcholine for different times in the absence of Na+, followed by determination of the carbamylcholine-induced increase in 22Na+ uptake (Fig. 3). The rate of 22Na+ uptake by the receptor declined to approximately 10 y. of the original value during a 5-min exposure to carbamylcholine in the absence of Na+, with 50% loss of activity in 30 s in this experiment.
A significant correlation between the rate of desensitization and the morphological maturity of the muscle cells was noted.
Cell cultures in which myotube formation was complete, but in which no further differentiation had occurred, had high levels of receptor transport activity but desensitized relatively slowly as in Figs of receptor Naf transport activity. Muscle cells were preincubated at 36" for the indicated times in assay medium containing 1 mM carbamylcholine and Tris+ rather than Na+. 22Na+ uptake was then measured for 30 s at 36" in normal medium containing 5 mM ouabain and 1 mM carbamylcholine. MINUTES FIG. 4. Effect of temperature on receptor Na+ transport activity. ezNa+ uptake was measured at 2', 21°, and 36" as described under "Experimental Procedure" in the presence of 5 mM ouabain and 5 mM carbamylcholine. zzNa+ uptake in the absence of carbamylcholine at each temperature has been subtracted from the results.
hypolemmal nuclei, and contracted spontaneously also had high levels of activity but desensitized more rapidly. In some experiments, complete desensitization occurred in 10 s. Since such rapid desensitization precluded accurate measurement of initial rates, conditions were sought which would slow desensitization. Removing extracellular Ca+ and adding ethylene glycol his@-aminoethyl ether)-N,N'-tetraacetic acid was partially effective (16), however, lowering the assay temperature proved to be more successful.
The initial rate of 22Na+ uptake catalyzed by the receptor is essentially temperature-independent between 2" and 36" (Fig. 4). At 2', however, the initial rate is maintained for a longer period of time. Rates of uptake remained constant for at least 20 s in all cultures tested at 2' (Fig. 4). Thus, although the initial rate of 2zNa+ uptake is temperature-independent, the rate and extent -I for 3 hours at 36" in assay medium (0) or assay medium with 5 mM ouabain (0) and then carbamylcholine-dependent zeNa+ uptake was determined at 2' as described under "Experimental Procedure." 2ZNa+ uptake in the absence of carbamylcholine has been subtracted.
of desensitization are temperature-dependent. Since the initial rate of 22Na+ uptake in mature cultures can be measured more accurately at 2" than at physiological temperature, most of the experiments described here have been performed at 2".
Dependence of Na+ Transport Activity of Acetylcholine Receptor on [Na+]in and [Nu+]..t---In tivo, the acetylcholine receptor catalyzes net transport of Na+ into the muscle cell. In order to compare the rate of net transport with the rate of Na+-Na+ exchange, the rate of 22Na+ uptake was measured under conditions of net Na+ influx as in Figs. 1 to 4 and under conditions of isotope exchange at equilibrium. The results presented in Fig. 5 show that the initial rate of 22Na+ uptake into muscle cells with low internal Na+ is 1.8-fold greater than the rate of uptake into cells that have been preincubated with ouabain for 3 hours to bring the internal Na+ concentration to 152 mM. In four such experiments, the ratio of net influx to exchange averaged 1.9 f 0.2. Control experiments confirmed that neither the number of receptors per cell (measured by toxin binding) nor the resting Na+ permeability of the cells was affected by the 3-hour exposure to ouabain. Thus, the rate of net influx exceeds the rate of exchange, demonstrating that the receptor does not catalyze an obligatory exchange diffusion reaction at a detectable rate.
The increased rate of 22Na+ uptake under conditions of net influx cannot be ascribed to an increased driving force due to the transmembrane potential (17). Although the resting potential of -40 millivolts1 (18) is large enough to account for a ratio of net influx to exchange of 1.9 (17), the transmembrane potential in the presence of 5 mM carbamylcholine should be in the range of 0 to -10 millivolts corresponding to a ratio of less than 1.15. The difference in rate can more likely be ascribed to a competitive inhibition of 22Na+ influx by internal unlabeled Na+ (see "Discussion").
The initial rate of carbamylcholine-dependent 22Na+ uptake at 2' varies with [Na+],,t in a manner suggesting saturation of the ion transport mechanism by Na+ (Fig. 6A). Similar results are obtained under conditions of net Na+ influx or under conditions of isotope exchange at equilibrium (Fig. 6A). Double reciprocal plots (Fig. 6B)  FIG. 6. Effect of [Na+].,,t on zzNa+ uptake. A, 22Na+ uptake in 30 s at 2" was determined in assay medium containing the indicated concentrations of Na+ plus 5 mM ouabain and 5 mM carbamylcholine.
Iso-osmotic media with the indicated Na+ concentration were prepared by replacing portions of the NaCl in the standard assay medium with an osmotically equivalent concentration of sucrose. 22Na+ uptake in the absence of carbamylcholine has been subtracted.
B, the data in A have been replotted in a double reciprocal plot. Cells were assayed after preincubation for 3 hours at 36" in assay medium (0) or assay medium with 5 mM ouabain ( l ). A, 22Na+ uptake was measured for 30 s in assay medium containing the indicated concentrations of acetylcholine at 2" (O), carbamylcholine at 36" (A), or carbamylcholine at 2" (0). 22Na+ uptake in the absence of carbamylcholine and acetylcholine has been subtracted. B, the data in A have been plotted on the logarithmic coordinates of Brown and Hill (19). slight but significant deviation from linearity on the double reciprocal plot at [Na+lO,t greater than 100 mM. Unfortunately, examination of Na+ concentrations is limited at present to concentrations less than 160 mM by the osmolarity of the cytoplasm. This precludes a study of transport at Na+ concentrations greater than the K,.
At 36", the dependence of 22Na+ uptake on [Na+lout was linear from 0 to 150 mM, showing no evidence of saturation.
If the ion transport mechanism is the same at 2' and 36", the K, for Na+ at 36" must be higher than at 2'.
Activation and Inhibition of Receptor by Cholinergic Ligands-The initial rate of 22Na+ uptake by the acetylcholine receptor provides a direct measure of the permeability change due to receptor activation and thus can be used to study receptor activation and inhibition by cholinergic ligands. Titration curves of activation by carbamylcholine at 2" and 36" and acetylcholine at 2" are presented in Fig. 7A. The apparent K, for activation by carbamylcholine is temperature-dependent increasing from 300 to 800 pM as the temperature is decreased from 36" to 2". Acetylcholine activates with a KA of approximately 40 pM at 2'. Carbamylcholine-dependent ezNa+ uptake was then measured for 30 s at 2" in the presence of 1 rnM carbamylcholine, 5 mM ouabain, and the indicated concentration of inhibitors. 22Na+ uptake in the absence of carbamylcholine has been subtracted.
Activation under all three of these sets of conditions is significantly cooperative.
In each case, the data, when plotted on the coordinates of Brown and Hill (19) (Fig. 7B), give a line with slope greater than 1 (carbamylcholine at 2', nH = 1.4; carbamylcholine at 36", nH = 1.9; acetylcholine at 2", nH = 2.0). Thus, as has been shown for the electroplax receptor (10,20), activation of the acetylcholine receptors of cultured muscle cells is a cooperative process.
Activation of receptor Na+ transport activity by 1 mM carbamylcholine was inhibited by n-tubocurarine and atropine with half-maximal inhibition at 4 and 100 pM, respectively, at 2" (Fig.  8). Similar results were obtained in experiments at 36". Inhibition by n-tubocurarine exhibited little if any cooperativity (7261 = 0.9 to 1.2).
Tetrodotoxin, which inhibits Na+ transport by the action potential Na+ ionophore of cultured chick muscle with a Kr = 11 nM (13), has no effect on acetylcholine receptor Na+ transport at 30 pM (Fig. 8), confirming the conclusion that the action potential Na+ ionophore does not contribute to the observed Na+ movements. Na+ Transport Activity of Single Receptor-Snake venom a-toxins have been shown to bind specifically to the acetylcholine receptors of cultured muscle cells (89) by inhibition studies using nicotinic cholinergic agonists. The experiment presented in Fig.  9 compares the binding of [1251]diiodo-cr-bungarotoxin to muscle cells with the inhibition of the receptor Na+ transport activity by the iodinated toxin.
The two titration curves coincide with 50% inhibition and binding at 1.6 nM toxin confirming the specificity of toxin binding.
Binding of a-bungarotoxin therefore provides a measure of the number of acetylcholine receptors per muscle cell and nNa+ uptake provides a measure of the number of Na+ ions transported into a muscle cell per mm. The quotient of these two measurements is the turnover number of a single receptor.
Data from experiments in which initial rates of 22Na+ uptake at 2" and extents of [1251]diiodo-cy-bungarotoxin binding at 36" were measured are summarized in Table I. The  1780   Y  I  I  I  I  I  3.2 X 10" f 0.6 X lo6 specific activity of the receptor under these conditions is 3.2 X lo6 Na+ ions/min/toxin binding site. Since Vm,, is approximately twice the rate measured at 150 mM Na+ (Fig. 6B), and since there are 3 a-toxin binding sites per molecule of purified acetylcholine receptor from eel electroplax (21, 22), the turnover number of the acetylcholine receptor Na+ ionophore indicated by these results is 2 X 10' Na+ ions per min per receptor.

DISCUSSION
Measurement of initial rate of 22Na+ uptake in the presence of carbamylcholine appears to provide a useful method of assay of acetylcholine receptor activity of cultured muscle cells. It has the advantage of providing a direct measure of the permeability response of the entire cell population to known concentrations of activator. Initial rates can be measured adequately in highly differentiated cultures at reduced temperatures (2"). Permeabilities calculated from 22Na+ uptake during either net uptake or isotopic exchange at equilibrium are similar, demonstrating that the rate of Na+ movement is not limited by fluxes of other ions. Initial studies of activation and inhibition of acetylcholine receptors of cultured muscle by cholinergic ligands indicate general similarity to receptors in eel electroplax (10,20) and adult neuromuscular junction (23, 24). The general approach described here should be useful in studies of other receptors mediating ion permeability changes such as the receptors for glutamate, aspartate, glycine, and y-aminobutyrate in the central nervous system.
Comparison of measurements of 22Na+ uptake and bungarotoxin binding have suggested a Vmax for Na+ transport by the acetylcholine receptor Na+ ionophore of 2 X 10' min-' at 2' (Table I). This value is much larger than the value of 3 x lo3 min-r derived from studies of 22Na+ efflux from electroplax membrane vesicles (10). A larger estimate of 3 X 10' min+ has been derived from a comparison of carbamylcholine-induced conductance and binding of a-toxin in whole electroplax (25). Also, analysis of electrical noise produced by acetylcholine at the neuromuscular junction (26,27) and in cultured muscle (28) has led to estimates of 0.3 to 0.9 x lo-lo mho for the conductance during a single noise-producing event. Equating this with the conductance of an acetylcholine receptor Na+ ionophore gives an estimate of 4 to 12 X lo8 min-l at -40 millivolts. The difference between the estimates derived using comparisons of toxin binding and steady state flux measurements (2 to 3 X 107 min-l) and those derived from noise measurements (4 to I2 X 10s min-I) may reflect the fraction of time that a given ionophore is active in the presence of activator. Thus the noise measurements estimate the instantaneous activity of a single active ionophore. On the other hand, the steady state flux measurements estimate the steady state activity of an ensemble of receptors. If the activity of a single receptor ionophore is a series of repetitive square pulses (27), at any time, only a fraction of the receptor ensemble is active. Thus, the steady state measurement must underestimate the instantaneous activity of an active receptor by a fraction that is equal to the proportion of the time that an individual ionophore is active. If this interpretation is correct, the acetylcholine receptor Na+ ionophore is active only 2 to 8% of the time, even in the presence of a saturating level of activator.
Mechanisms of ion transport can be broadly divided into two classes: those involving a mobile carrier and those involving specific channels or pores. A major feature distinguishing these two classes of mechanisms is the extent to which macromolecular membrane components must move during transport of each substrate molecule. Thus, a mobile carrier must traverse or rotate through the membrane with each substrate molecule transported, while a channel must move during "opening" or activation but not during transport of each substrate molecule. These considerations predict that the rate of transport by a carrier should be: (a) relatively slow since large macromolecular components must move during transport (less than 6 X lo5 min+ (29)) and (b) highly temperature-dependent, especially at the phase transition of the membrane lipids, since the viscosity of the membrane in which the carrier must move is highly temperature-dependent. These limitations need not apply to a channel.
Experimental support for these distinctions between carrier and channel mechanisms has been provided by experiments on ion-conducting antibiotics. Transport by valinomycin and nonactin which behave as mobile carriers (Ref. 30 and references therein) is highly temperature-dependent, especially at the lipid phase transition (30), and relatively slow (6 x lo5 min+) (31) while transport by gramicidin, a channel-former (Ref. 30 and references therein), is temperature-independent (30) and relatively rapid (6 X lo* min-I) (32). Other channel-forming antibiotics have comparably high turnover numbers.
Most of the data available concerning transport in biological systems suggest the involvement of carriers (reviewed in Ref. 33). Large temperature dependences (33), especially at lipid phase transitions (3436), and relatively slow rates (less than lo5 min-1) (29) have been reported for transport of a number of metabolites 1781 in bacteria and for sugars and ions in mammalian erythrocytes. In addition to the characteristics of carriers described above, biological carrier systems exhibit transacceleration (33) and exchange diffusion, i.e. transport in which the rate of tracer influx is increased by increasing the internal substrate concentration (33).
The results described here provide three pieces of evidence indicating that the transport of Na+ by the acetylcholine receptor Na+ ionophore is best described by a channel or pore mechanism: (a) the transport process is essentially temperatureindependent between 2" and 36", (b) the rate is more rapid (2 X 107 min-1) than the limiting transport rate of even low molecular weight ion carriers such as valinomycin, and (c) the rate of uptake is not increased by increasing the internal Na+ concentration indicating that exchange diffusion does not contribute significantly to the observed transport. The action potential Na+ ionophore also appears to transport Na+ via a channel mechanism since the rate of ion movement (lo* mini) is too large to be accommodated by a carrier mechanism (37) and ion transport is not highly temperature-dependent (17). These two transport systems appear to be unique among biological systems studied to date.
Although the acetylcholine receptor appears to transport Na+ via a channel mechanism, the transport process does show saturation at 2". While saturability is more often considered a property of carrier systems, channel mechanisms that incorporate saturation have been formulated (39, and the rate of ion transport by some channel-forming antibiotics has a high concentration limit (32). The results of Fig. 6 suggest that a binding site for Na+ exists within the receptor Na+ channel which at 2" interacts with Naf during the rate-limiting step in transport in a manner described approximately by the Michaelis-Menten relation with a K, of 150 mM. If a similar interaction occurs at physiological temperature, the K, must be higher. The binding site within the channel suggested by these data might play a role in determining the selectivity of ion transport.
The presence of a Na+ binding site within the receptor Naf channel leads to the possibility of competitive inhibition of Na+ transport by Na+ and other transported ions. In particular, the observed inhibition of %a+ influx by internal Na+ (Fig. 5) is expected.
Internal and external Na+ ions would have equal probability of occupying a Na+ binding site symmetrically oriented within the receptor Na+ channel.
Since occupancy by internal Na+ would not result in 22Naf uptake, the rate of zzNa+ uptake would be reduced 2-fold under conditions of isotope exchange.
These considerations suggest that a detailed analysis of transport rates in the presence of known internal and external ion concentrations might provide considerable insight into the interaction of ions with the acetylcholine receptor Na+ channel during transport.