Mutations in the M4 domain of the Torpedo californica nicotinic acetylcholine receptor alter channel opening and closing.

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type alpha418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the beta subunit (beta447Trp) had similar but smaller effects on mean open time. Coexpression of alpha418Trp and beta447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the alpha418Trp and beta447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from alpha418Trp mutants. Burst analysis of the alpha418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the alpha- and beta subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time.

Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction.

The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, epsilonL269F, found in a family with SCCMS, using both in vitro and in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro, similar to the phenotype of other reported mutants. Transgenic mice expressing this mutant develop a syndrome that is highly reminiscent of the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal accumulation of calcium and striking ultrastructural changes, including enlargement and degeneration of the subsynaptic mitochondria and nuclei. These findings clearly demonstrate the role of this mutation in the spectrum of abnormalities associated with the SCCMS and point to the subsynaptic organelles as principal targets in this disease. These transgenic mice provide a useful model for the study of excitotoxic synaptic degeneration.

Mouse-Torpedo chimeric alpha-subunit used to probe channel-gating determinants on the nicotinic acetylcholine receptor primary sequence.

1. To determine if structural domains are important for nicotinic acetylcholine receptor (nAChr) channel function, six mouse-Torpedo chimeric alpha-subunits were constructed (Fig. 2) and coexpressed with Torpedo californica beta-, gamma-, and delta-subunits in Xenopus laevis oocytes. 2. nAChRs containing a chimeric alpha-subunit were examined by voltage- and patch-clamp methods to determine their functional characteristics. Dose-response curves from voltage-clamped oocytes were used to estimate EC50's and Hill coefficients. Whole-cell currents were normalized against the alpha-bungarotoxin (alpha-BTX) binding sites to obtain normalized responses to acetylcholine (ACh). Open time constants at 4 microM ACh were used to examine single-channel behavior. 3. The EC50 for ACh was modulated by the N-terminal half of the alpha-subunit. When the Torpedo subunit sequence between position 1 and position 268 was replaced by mouse sequence, the EC50 shifted toward the value for the wild-type mouse subunit. Replacement of either the 1-159 or the 160-268 positions of the Torpedo sequence with the mouse sequence lowered the EC50. This suggests that at least two regions play a role in determining the EC50. 4. When the primary sequence (160-268) of the Torpedo alpha-subunit was introduced in the mouse alpha-subunit (T160-268), the expressed chimeric receptor was nonfunctional. The inverse chimera (M160-268) was functional and the open time constant and EC50 were similar to those of mouse but the normalized response was characteristic of Torpedo. 5. The normalized macroscopic response to ACh (300 microM) of the chimera containing the mouse alpha-subunit showed a ninefold increase relative to the Torpedo wild type. Receptors which contain the C terminal of the mouse alpha-subunit also show an increase in the maximum normalized current. Receptors with the alpha-subunit which contain the Torpedo C-terminal sequence have a lower normalized response. 6. The combined results suggest that AChR channel function is modulated by structural determinants within the primary sequence. These structural domains might modulate channel function through specific allosteric interactions. The lack of response of the T160-268 chimera suggests that a critical interaction essential for the coupling of agonist binding and channel gating was disrupted. This result suggests that the interaction of structural domains within the nAChR primary structure are essential for channel function and that these intractions could be very specific within different nAChR species.

Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating.

Our previous amino acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha C418 position. A tryptophan substitution on the alpha C418 produced a 3-fold increase in normalized macroscopic response to acetylcholine in voltage-clamped Xenopus laevis oocytes (Lee et al., 1994). This result was explained by a 23-fold decrease in the closing rate constant measured from single-channel analysis (Ortiz-Miranda et al., 1996). In this study, we introduce more tryptophan substitutions at different positions of this postulated lipid-exposed segment M4 in order to examine functional consequences at the single-channel level. From a series of amino acid substitutions at alpha G421, only phenylalanine and tryptophan produced a substantial increase in the open time constant. The lack of response from a tyrosine substitution at the alpha G421 suggests that the side chain volume is not the main structural element responsible for the effect of tryptophan on the stabilization of the open state of the channel. Three multiple mutants, alpha C418W/G421A, alpha C418W/G421W, and alpha C418W/beta C447W, were constructed in order to establish the correlation between the number of lipid-exposed tryptophans and the channel open time constant. The alpha C418W/G421A double mutant demonstrated that when both previous mutations are combined the open time constant was increased 1.5-fold relative to the alpha C418W. When the two mutants (alpha C418W and alpha G421W) were combined in a single mutation, a functional receptor was expressed and the open time constant of the new double mutant increased to 33.4 ms, an 80-fold increase relative to wild type. Estimations of free energy changes calculated from the rate constant for the opening transition suggest that each tryptophan contributes to the stabilization of the open state of the channel by about 0.8 kcal/mol, and the effect of tryptophan substitutions on the free energy is additive. This result suggests that in the channel gating mechanism of the AChR, each subunit contributes independently to the energy barrier between the open and closed state. At selected positions within the postulated lipid surface of the AChR, tryptophan substitutions could establish hydrophobic and perhaps dipole interactions that may play a dramatic role in the channel gating mechanism.

Effects of antibody binding on structural transitions of the nicotinic acetylcholine receptor.

Patch-clamping and photoaffinity-labeling techniques were used to study the effects of binding of monoclonal antibodies (mAbs) on the function of Torpedo californica nicotinic acetylcholine receptor (nAChR). The rat anti-Torpedo nAChR mAbs examined here are known to inhibit ligand binding to either the high-affinity (mAb 247) or both the high- and low-affinity binding sites (mAb 370 and mAb 387) [Mihovilovic, M. & Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059; Mihovilovic, M., & Richman, D. P. (1987) J. Biol. Chem. 262, 4978-4986]. Single-channel analysis shows that mAb 247 and the Fab fragment of mAb 247 inhibit the opening of the nAChR ion channel, although they have no effects on the structural transition from the resting to desensitized state as monitored by the extent of decreased labeling by the photoreactive probe 3-(trifluoromethyl)-3-(m- [125I]iodophenyl)diazirine ([125I]-TID). In the presence of mAb 387, the nAChR single-channel amplitude was decreased by 20%, whereas Fab 387 completely inhibited channel opening. [125I-TID]-labeling studies suggest that the mAb 387-nAChR and Fab 387-nAChR complexes are able to undergo the transition between resting and desensitized states. This result confirms that the nAChR can assume a desensitized state without prior channel opening. In addition, mAb 35 and mAb 132, which recognize the main immunogenic region (MIR) of the nAChR, and mAb 370 do not alter either single-channel behavior or labeling patterns. Combining the results from characterization with respect to their epitopes and their effects on agonist (carbamylcholine) and antagonist [alpha-bungarotoxin (alpha-BTX) and curare] binding, these results indicate that mAbs could be used to map functional and structural domains.

Differential desensitization properties of rat neuronal nicotinic acetylcholine receptor subunit combinations expressed in Xenopus laevis oocytes.

1. Chronic administration of nicotine up-regulates mammalian neuronal nicotinic acetylcholine receptors (nAChRs). A key hypothesis that explains up-regulation assumes that nicotine induces desensitization of receptor function. This is correlated with behaviorally expressed tolerance to the drug. 2. The present experiments were conducted to: (a) obtain information on the nicotine-induced desensitization of neuronal nAChR function, a less understood phenomenon as compared to that of the muscle and electric fish receptor counterparts; (b) test the hypothesis that different receptor subunit combinations exhibit distinct desensitization patterns. 3. Xenopus laevis oocytes were injected with mRNAs encoding rat receptor subunits alpha 2, alpha 3, or alpha 4 in pairwise combination with the beta 2 subunit. The responses to various concentrations of acetylcholine (ACh) or nicotine were analyzed by the two electrode voltage clamp technique. 4. Concentration-effect curves showed that nicotine was more potent than ACh for all the receptor subunit combinations tested. Only the alpha 4 beta 2 combination exhibited a depression of the maximum effect at concentrations higher than 20 microM nicotine. 5. After a single nicotine pulse, receptor desensitization (calculated as a single exponential decay) was significantly slower for alpha 4 beta 2 than for either alpha 3 beta 2 or alpha 2 beta 2. 6. Concentrations of nicotine that attained a near maximum effect were applied, washed, and re-applied in four minute cycles. The responses were calculated as percentages of the current evoked by the initial application. Following 16 minutes of this protocol, the alpha 4 beta 2 combination showed a greater reduction of the original response as compared to the alpha 2 beta 2 and alpha 3 beta 2 subunit combinations. Taking points 5 and 6 together, these experiments suggest that the alpha 4 beta 2 receptor subtype desensitizes at a slower rate and remains longer in the desensitized state. 7. Because alpha 4 beta 2 is the main receptor subunit combination within the brain and is up-regulated by nicotine, our data may be important for understanding the molecular basis of tolerance to this drug.

Heterogeneous distribution of acetylcholine receptors in chick myocytes induced by cholesterol enrichment.

The cholesterol concentration at the cell surface of cultured chick myocytes was increased in order to determine the effects of high levels of cholesterol on the ion channel properties of the nicotinic acetylcholine receptor. Single channel recordings and fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed under equivalent conditions for normal and cholesterol enriched myocytes. In cell attached patches from myocytes with a cholesterol to phospholipid molar ratio (c/p) of 0.24 and a microviscosity of 1.35 poise a single conductance of 51 pS was detected. The cholesterol enriched myocytes with a c/p of 0.52 and a microviscosity of 2.05 poise showed two conductances, a 54 pS and a 39 pS channel: both were blocked by alpha-bungarotoxin. The 39 pS channel was detected with the simultaneous appearance of a slow component of tau m (modulation time) for DPH fluorescence measured by phase demodulation. The 80% reduction in the open time constant (tau 2) of the 39 pS channel suggest an inhibition of the normal conformational state. The combined results suggest that cholesterol enrichment may induced a more heterogeneous lipid environment and that the two types of channel properties could result from the distribution of the receptors in different domains.

Modulation of acetylcholine receptor channel by a polar component isolated from toxic Ostreopsis lenticularis extracts.

Methanol extracts obtained from O. lenticularis clones are toxic to mice and inhibit acetylcholine-induced contractions in frog skeletal muscle. Chromatographic fractionation of extracts produced two major fractions with different retention times. Single channel recordings in myocyte membrane patches exposed to more polar fraction showed the appearance of acetylcholine-activated channels whose mean current amplitude was nearly half that of the controls. Channel open times under control and experimental conditions were similar. Thus, this dinoflagellate fraction reduces the ionic conductance of nicotinic receptor channels without altering their lifetime.

Positive modulators of muscle acetylcholine receptor.

A solution of succinic anhydride (SA) in buffer N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES), the SATES solution, potentiates the effect of carbonyl containing agonists on frog muscle (del Castillo et al., Br. J. Pharmac. 84: 275-288, 1985). Here we report that the main compound in the SATES solution is a monosuccinyl ester of TES (MST). This compound is not an agonist of the acetylcholine (ACh) receptor nor is it an inhibitor of ACh esterase, yet MST potentiates the ACh-induced tension and depolarization in frog muscle to a new maximum. It increases the amplitude of miniature endplate potentials but has no effect on the time course of miniature endplate currents. The acetylated analogue of MST (mono-acetyl-TES: MAT) had similar but more pronounced effects on frog muscle. Neither MST nor MAT affect [3H]Ach binding to receptor-rich membranes from the electric organ of Torpedo californica, or the affinity state transition induced by agonists. Radiolabeled MST does not bind to these membranes and MAT does not alter agonist-induced ion flux. Therefore, these compounds seem to act as positive modulators of muscle ACh receptor but are inactive in Torpedo vesicles.