Two distinct nicotinic receptors, one pharmacologically similar to the vertebrate alpha7-containing receptor, mediate Cl currents in aplysia neurons

Abstract

Ionotropic, nicotinic receptors have previously been shown to mediate both inhibitory (Cl-dependent) and excitatory (cationic) cholinergic responses in Aplysia neurons. We have used fast perfusion methods of agonist and antagonist application to reevaluate the effects on these receptors of a wide variety of cholinergic compounds, including a number of recently isolated and/or synthesized alpha toxins [alpha-conotoxin (alphaCTx)] from Conus snails. These toxins have been shown in previous studies to discriminate between the many types of nicotinic receptors now known to be expressed in vertebrate muscle, neuroendocrine, and neuronal cells. One of these toxins (alphaCTx ImI from the worm-eating snail Conus imperialis) revealed that two kinetically and pharmacologically distinct elements underlie the ACh-induced Cl-dependent response in Aplysia neurons: one element is a rapidly desensitizing current that is blocked by the toxin; the other is a slowly desensitizing current that is unaffected by the toxin. The two kinetically defined elements were also found to be differentially sensitive to different agonists. Finally, the proportion of the rapidly desensitizing element to the sustained element was found to be cell-specific. These observations led to the conclusion that two distinct nicotinic receptors mediate Cl currents in Aplysia neurons. The receptor mediating the rapidly desensitizing Cl-dependent response shows a strong pharmacological resemblance to the vertebrate alpha-bungarotoxin-sensitive, alpha7-containing receptor, which is permeable to calcium and mediates a rapidly desensitizing excitatory response.

Fig. 1.

Cl-dependent responses as a function of holding potential in three different cell types: 2 sec application of 200 μm ACh. Comparing A (posterior neuron and pleural ganglion), B (buccal cell 10), andC (buccal cell 3), it can be seen that for different cell types the Cl-dependent response shows markedly different desensitization kinetics over the 2 sec application of ACh. In all three cells, the response on one side of E_Clis the mirror image of the response on the other side ofE_Cl; the dashed linesrepresent the responses measured in the three cells at −70, −75, and −70 mV, respectively, which have been inverted and superimposed on the responses taken at holding potentials less negative than but equidistant from E_Cl (solid lines).

Fig. 2.

Selective elimination by αCTx ImI of the rapidly desensitizing Cl-dependent response in identified neurons of the buccal and pleural ganglia (see Materials and Methods). A, αCTx ImI (1 μm) included in the ACh tube had no effect on the response (compare response presented as a solid line with that presented as an enhanced dashed line). The same concentration of αCTx ImI added to the control tube (hence bathing the cell before the ACh application), however, selectively blocked the desensitizing element of the response.B, In another cell of the same type, increasing concentrations of αCTx ImI failed to significantly alter the sustained element of the Cl-dependent response to 250 μmACh applied at 1 min intervals (records in toxin taken after a 4 min exposure to each concentration). C, In a neuron (posterior cell from the pleural ganglion; Kehoe, 1972a), which shows only the rapidly desensitizing element of the Cl-dependent response, 1 μm αCTx ImI completely blocks the response to 200 μm ACh. D, E, Selective block by 1 μm αCTx ImI of the rapidly desensitizing element of the response of two identified neurons of the buccal ganglion (B10 and B3, respectively) to 200 μm ACh. The two cells show different, characteristic proportions of the rapidly desensitizing and the sustained elements.

Fig. 3.

Progressive diminution in the rapidly desensitizing element of the Cl-dependent response of identified buccal cells with increasing concentrations of αCTx ImI. In all experiments, 200 μm ACh was applied at 1 min intervals, and increasing concentrations of αCTx ImI were introduced successively in the control tube of the fast perfusion system. The cells were exposed to a continual flow of each concentration for ∼3–4 min. A, Records from a buccal cell having only a very weak sustained element.B, Records from a buccal cell with a prominent sustained element. The response in 1 μm αCTx ImI was subtracted from the response at each of the other concentrations (dashed lines) to obtain records of only the rapidly desensitizing, toxin-sensitive responses (solid lines).C, Data from eight experiments were used to calculate the average reduction of the desesensitizing element of the control response with increasing concentrations of αCTx ImI (10, 20, 50, 100, and 500 nm and 1 μm). For cells having both rapidly desensitizing and sustained responses, the amplitude of the rapidly desensitizing element was obtained by subtracting the response obtained in 1 μm toxin from that obtained at each of the other concentrations. Because identical concentrations of toxin were not used in each experiment, the n values have been indicated in the inset. The SEs have been indicated for each concentration, except for 500 nm, for which only one point was obtained, and for 1 μm, which yielded a total block (either by definition, see above, or by direct reading of the data) of the desensitizing response in all cases. The average IC₅₀ for the eight experiments illustrated here was estimated by interpolation to be 47 nm.

Fig. 4.

A–E, Failure of MLA, αBTx, dβe, strychnine, and TC to discriminate between the two elements of the Cl-dependent response of buccal cells to 200 μm ACh. The data illustrated were obtained from five different cells. ACh (200 μm) was applied at 1 or 2 min intervals, and the antagonists were applied, at increasing concentrations, exclusively through the control tube. With the exception of 10 nm MLA, all concentrations of all five antagonists tested affected both elements of the Cl-dependent response.A′–B′, Comparison in the same cell of the block by 50 nm MLA and 50 nm αBTx of the rapidly desensitizing and sustained components of the two-component Cl-dependent response in buccal neurons elicited by a 2 sec application of 200 μm ACh. A complete recovery from the block by MLA was obtained before applying αBTx. C′–E′, Comparison in the same cell of the block by 20 μm dβe, 20 μm strychnine, and 20 μm TC of the rapidly desensitizing and sustained components of the Cl-dependent response elicited in a buccal neuron by a 2 sec application of 200 μm ACh. The three different antagonists were tested successively on the same cell. A thorough wash, resulting in the total recovery of the initial response amplitude, separated the three experiments. In A′–E′, the blocked responses, obtained by subtraction, are represented by dashed lines.

Fig. 5.

Preferential activation, by cytisine and D6, of the rapidly desensitizing and sustained Cl-dependent responses, respectively. A, Comparison, in a buccal cell showing a marked sustained element in the ACh Cl-dependent response, to 100 μm ACh, 10 μm D6, and increasing concentrations of cytisine (10, 50, and 100 μm). Note that at the concentrations applied here, there is practically no evidence of a sustained Cl-dependent response to cytisine, and that the response to 10 μm cytisine (4.54 nA) is equal in amplitude to the rapidly desensitizing response to 100 μmACh (4.53 nA, obtained by subtraction of the sustained element from the total ACh response). For easier evaluation of the cytisine-induced currents, they have been reproduced in the inset with an expanded x-axis and a reduced y-axis.B, Responses of a similar buccal cell to 100 μm ACh and to increasing concentrations of suberyldicholine (1, 10, and 100 μm). Note that the sustained response to 10 μm suberyldicholine is larger than that to 100 μm ACh. C, Responses to increasing concentrations of both cytisine (C′; cytisine and ACh records presented in inset with expandedx-axis and reduced y-axis) and suberyldicholine (C") of a buccal cell showing a predominantly rapidly desensitizing response to 100 μmACh (C′ and C"). Note that the responses to all three agonists desensitize during the 2 sec application, and that the response to 10 μm cytisine (C′) is larger than that to 100 μm ACh, whereas no response can be detected to 10 μm suberyldicholine (C"). The desensitizing response to 100 μmsuberyldicholine (C") is only approximately one-third that of the response to the same concentration of ACh.

Fig. 6.

Further evidence for the differentiation, by suberyldicholine and cytisine, of two distinct Cl-dependent responses.A, Effect of 500 nm αCTx ImI on the responses to 100 μm ACh and 20 μmsuberyldicholine of a buccal cell showing a prominent sustained element in the two-component Cl-dependent response. Note the selective elimination in the ACh response of the rapidly desensitizing element and a failure of the toxin to affect the response to suberyldicholine. The periodic rapid deflections seen in the ACh records inA represent spontaneous synaptic activity.B, Effect of 1 μm cytisine (applied in the control tube only) on the ACh response of a buccal cell to 100 μm ACh and 10 μm suberyldicholine. Cytisine can be seen to block only the rapidly desensitizing element of the ACh response, whereas it has no effect on the response to suberyldicholine.

Fig. 7.

Effect of C6 and αCTx ImI on the ACh-induced cationic response. ACh was applied on the cell soma for 2 sec once every minute (A, B) or once every 90 sec (C). Between each ACh application, the holding potential was alternately set at −30 or −90 mV. A, C6 (20 μm) present in both the control and agonist tubes blocks the cationic response to 200 μm ACh only when the ligand-gated channel is open, and it does so in a voltage-dependent manner. The records taken in the presence of C6 are superimposed on the control records in the right column (control vs C6). Whereas at −30 mV the responses are essentially identical in the absence (control, solid line) and presence (C6, dashed line) of the antagonist, at −90 mV, a reduction in amplitude rapidly develops during the 2 sec application. Note, however, that even at −90 mV the response amplitude at the beginning of the 2 sec application is unaffected by the presence of C6. These records were taken after 5 min exposure to C6 through the control tube. B. αCTx ImI (1 μm) blocks the cationic response to 250 μm ACh in a non-voltage-dependent manner. The toxin, flowing continuously and only from the control tube, reached its maximum blocking effect within 3 min. The records shown in the middle column were taken after ∼7 min exposure to αCTx ImI. In the right column, the control records have been superimposed on the records taken in the presence of toxin, which have been multiplied by 7.2. At both potentials, the amplified toxin records superimpose directly on the control responses, showing that there was no voltage-dependent element in the toxin block of the cationic ACh response. C, Progressive and reversible block of the cationic response to 250 μm ACh with increasing concentrations of αCTx ImI, with each increasing toxin concentration being placed in the control tube after the stabilization of the response amplitude at the previous toxin concentration. Ten minutes after returning toxin-free ASW to the control tube, the response returned to its control amplitude.

Fig. 8.

Effect of αCTx MII, αA-CTx EIVA, αCTx PnIA, and αCTx PnIB on the cationic and Cl-dependent responses to 100 or 200 μm ACh. A, B, Neither αCTx MII (1 μm) nor αA-CTx EIVA (1 μm) has an effect on either the cationic response of the unpigmented pleural ganglion cells (A, B, left records) or on either component of the Cl-dependent responses in buccal neurons (A, B, right records). C, αCTx PnIA (1 μm) weakly blocks the cationic response as well as the sustained Cl-dependent response (left and right columns, respectively) and strongly blocks the rapidly desensitizing Cl-dependent response in the same cell (right column). D, αCTx PnIB (1 μm) causes a small reduction in all three nicotinic responses (cationic, rapidly desensitizing, and sustained Cl-dependent responses). No agonist effect of any of the four toxins could be detected.

Fig. 9.

Relative ability of eight antagonists to block the three types of nicotinic response to 200 μm ACh expressed in approximate IC₅₀ values. Estimates of IC₅₀values were made by visual inspection of the data and have been established with the notion of obtaining a weighted rank ordering rather than of obtaining rigorously defined IC₅₀ values (see Results). A perusal of a given tone of column (black, gray, or white) permits an evaluation of the differential facility with which the different antagonists block a given response type. A comparison of the three column tones for each antagonist reveals the ability of a given antagonist to discriminate between the different response types. Empty columns with two asterisks are cases in which no effect (NE) on the ACh response was seen with the maximum concentration tested, the value of which is indicated in lieu of the data column. One asterisk indicates that the antagonist can induce a slight reduction in the tagged response, but that the highest concentration used failed to block the response by half (only case being that of 20 μm αCTx ImI on the sustained Cl response). V-d, Instances in which the block of the cationic response was voltage-dependent.

Fig. 10.

Relative effectiveness of different cholinomimetics in eliciting the three different types of nicotinic response. The log scale on which the data have been plotted shows, for example, that under the criteria used here (see Results), nicotine and cytisine are at least 10 times more effective than ACh in eliciting the rapidly desensitizing Cl-dependent response, whereas DMPP is 20 times less effective. Because of the desensitizing block (D-b) that nicotine and cytisine induce in the desensitizing Cl response, only low concentrations (1–20 μm) of those agonists were used in determining their relative efficacy on that receptor. The double asteriskindicates cases in which no response at all could be elicited (NE, no effect) with the highest concentration tested (200 μm).