Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor.

Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (gamma M3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC(50), is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized alpha C418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the gamma M3/alpha M4 double mutants, the EC(50)s were similar to those measured for the alpha C418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs.

Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

Previous amino acid substitutions at the M4 domain of the Torpedo californica and mouse acetylcholine receptor suggested that the location of the substitution relative to the membrane-lipid interface and perhaps to the ion pore can be critical to the channel gating mechanism [Lasalde, J. A., Tamamizu, S., Butler, D. H., Vibat, C. R. T., Hung, B., and McNamee, M. G. (1996) Biochemistry 35, 14139-14148; Ortiz-Miranda, S. I., Lasalde, J. A., Pappone, P. A., and McNamee, M. G. (1997) J. Membr. Biol. 158, 17-30; Tamamizu, S., Lee, Y. H., Hung, B., McNamee, M. G., and Lasalde-Dominicci, J. A. (1999) J. Membr. Biol. 170, 157-164]. In this study, we introduce tryptophan substitutions at 12 positions (C412W, M415W, L416W, I417W, C418W, I419W, I420W, G421W, T422W, V423W, S424W, and V425W) along this postulated lipid-exposed segment M4 so that we can examine functional consequences on channel gating. The expression levels of mutants C412W, G421W, S424W, and V425W were almost the same as that of the wild type, whereas other mutants (M415W, L416W, C418W, I419W, I420W, T422W, and V423W) had relatively lower expression levels compared to that of the wild type as measured by iodinated alpha-bungarotoxin binding ([(125)I]-alpha-BgTx). Two positions (L416W and I419W) had less than 20% of the wild type expression level. I417W gave no detectable [(125)I]BgTx binding on the surface of oocyte, suggesting that this position might be involved in the AChR assembly, oligomerization, or transport to the cell membrane. The alphaV425W mutant exhibited a significant increase in the open channel probability with a moderate increase in the macroscopic response at higher ACh concentrations very likely due to channel block. The periodicity for the alteration of receptor assembly and ion channel function seems to favor a potential alpha-helical structure. Mutants that have lower levels of expression are clustered on one side of the postulated alpha-helical structure. Mutations that display normal expression and functional activity have been shown previously to face the membrane lipids by independent labeling studies. The functional analysis of these mutations will be presented and discussed in terms of possible structural models.

Alteration in ion channel function of mouse nicotinic acetylcholine receptor by mutations in the M4 transmembrane domain.

The effect of structural alterations of the M4 transmembrane segment in the Torpedo californica AChR has shown that substitution of specific residues can be critical to the channel gating (Lasalde et al., 1996). In a previous study we found that phenylalanine and tryptophan substitutions at the alphaC418 residue in the M4 transmembrane segment of the Torpedo californica AChR significantly altered ion channel function (Lee et al., 1994; Ortiz-Miranda et al., 1997). Cassette mutagenesis was used to mutate the Cys residue at the corresponding C418 position in the alpha subunit of mouse AChR. A total of nine mutations on the mouse alphaC418 position were tested, including the alphaC418A, alphaC418V, alphaC418L, alphaC418S, alphaC418M, alphaC418W, alphaC418H, alphaC418E and alphaC418G mutants. All the mutants tested were functional except the alphaC418G which was not expressed on the surface of the oocyte. The data obtained from macroscopic and single channel currents demonstrate that different types of amino acids can be accommodated at this presumably lipid-exposed position without loss of ion-channel function. As with the Torpedo AChR, the mutation of Cys to Trp dramatically decreased the EC(50) for acetylcholine and increased channel open time. The lack of expression of the mouse alphaC418G suggest that there are some differences in folding, oligomerization and perhaps transport to the surface membrane for this mutant between the Torpedo and the mammalian AChR.

Topological disposition of Cys 222 in the alpha-subunit of nicotinic acetylcholine receptor analyzed by fluorescence-quenching and electron paramagnetic resonance measurements.

The structure of the nicotinic acetylcholine receptor (AChR) has been studied using a combination of fluorescence quenching and electron paramagnetic resonance (EPR) collision gradient methods. The AChR from Torpedo californica was labeled with a fluorescent probe, N-(1-pyrenyl)maleimide, specific for sulfhydryls in a hydrophobic environment, under conditions of selective labeling of Cys222 in the alpha-subunit. alphaCys222 is located in the postulated M1 transmembrane domain and predicted to be at the center of an alpha-helical secondary structure. The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by fluorescence quenching measurements. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, suggesting that the fluorophore is located closer to the membrane-water interface than to the hydrophobic interior. Power saturation EPR spectroscopy was also used to examine the effect of molecular oxygen and water-soluble paramagnetic reagents on the saturation behavior of a nitroxide spin label, which was specifically attached to the same alphaCys222 residue. Using the gradients of these paramagnetic reagents through the membrane-solution interface, the distance for the nitroxide derivative from the membrane-solution interface was measured to be approximately 7 A from the headgroup region of the phospholipid bilayer, in agreement with fluorescence quenching results. These results suggest that the M1 transmembrane domain of the AChR probably forms an irregular structure, a beta-strand, or an alpha-helical structure that may span the membrane in a way different from a linear alpha-helix.

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.

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.

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine.

1. Site directed mutagenesis was used to alter the structure of Torpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed in Xenopus laevis oocytes injected with in vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp. 2. A series of mutations of a highly conserved Arg at position 209 of the alpha subunit of Torpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (alpha R209K) or His (alpha R209H) at position 209 shifted the EC50 for ACh slightly from 5 microM to 12 microM and increased the normalized maximal channel activity 8.5- and 3.2-fold, respectively. 3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition. 4. Further mutations at different positions of the alpha subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutations alpha I210A and alpha L212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine. 5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be beta sheet structure.

Lipid modulation of nicotinic acetylcholine receptor function: the role of membrane lipid composition and fluidity.

The effects of membrane lipid composition and fluidity on AChR ion channel function were studied after reconstituting the receptor with sphingomyelin, phosphatidylcholines with different degrees of unsaturation, or different neutral lipids. AChR ion flux activity was shown to be retained in some membranes of both high and low fluidity, as measured by the steady-state anisotropy of the membrane probes diphenylhexatriene and trimethylammonium diphenylhexatriene. The results suggest that lipid composition is more important than bulk membrane fluidity in determining AChR ion channel function.

Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function. 2. Membrane fluidity and ligand-mediated alteration in the accessibility of gamma subunit cysteine residues to cholesterol.

Fluorescence-quenching and energy-transfer measurements were carried out to further characterize lipid-protein interactions involving the nicotinic acetylcholine receptor (AChR) from Torpedo californica in reconstituted membranes. To assess the fluidity of the receptor microenvironment, cis- and trans-parinaric acids were used to take advantage of the preferential partitioning behavior of the trans isomer for the gel phase. A relatively higher extent of energy transfer from the intrinsic tryptophan fluorescence of AChR in dielaidoylphosphatidylcholine bilayers to cis-parinaric acid in both the gel and the fluid phase suggests that the AChR is surrounded by a relatively fluid annulus of lipids. The ability of AChR to accommodate and interact with specific lipids such as cholesterol and fatty acids in the vicinity of pyrene-labeled cysteine residues in the membranous domain and/or the membrane-water interface region of the gamma subunit was assessed. Pyrene-labeled AChR prepared in (6,7-dibromostearoyl)phosphatidylcholine showed a 25% decrease in fluorescence as sites accessible to phospholipids were occupied; subsequent addition of dibromocholesterol hemisuccinate (DiBrCHS) caused further quenching by about 25%. This result is consistent with the presence of sites accessible to cholesterol, but not accessible to phospholipids, in the vicinity of the cysteine-bound pyrene in the membranous domain of the AChR. Quenching by DiBrCHS was sensitive to the presence of an AChR activator (carbamylcholine) but not a competitive antagonist (alpha-bungarotoxin). The Stern-Volmer quenching constant was 0.123 in the absence of added ligands and 0.167 and 0.134 in the presence of carbamylcholine and alpha-bungarotoxin, respectively, corresponding to accessibilities of 65%, 90%, and 70%.

Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function. 1. Spatial disposition of cysteine residues in the gamma subunit analyzed by fluorescence-quenching and energy-transfer measurements.

The nicotinic acetylcholine receptor from Torpedo californica was labeled with a fluorescent, lipophilic probe, N-(1-pyrenyl)maleimide, specific for sulfhydryls in a hydrophobic environment, and was found to alkylate Cys 416, Cys 420 and Cys 451 in the gamma subunit [Li, L., Schuchard, M., Palma, A., Pradier, L., & McNamee, M.G. (1990) Biochemistry 29, 5428-5436]. The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by a combination of fluorescence-quenching and resonance energy transfer measurements, under conditions of selective labeling of the gamma subunit. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, with a Stern-Volmer quenching constant of 0.187 compared to 0.072 for the latter, suggesting that the fluorophore(s) are located closer to the membrane-water interface rather than the hydrophobic interior. Energy transfer was found to occur from tryptophan in the acetylcholine receptor to cysteine-bound pyrene with a distance of separation of approximately 18 A. However, there was no energy transfer when pyrene-labeled AChR was reconstituted into membranes containing brominated phospholipids and cholesterol, suggesting that the fluorophore(s) responsible for energy transfer are located in the membrane domain. Thus, the N-(1-pyrenyl)maleimide can be used to monitor lipid-protein interactions of the AChR.

FTIR analysis of nicotinic acetylcholine receptor secondary structure in reconstituted membranes.

Using Fourier-transform infrared resonance spectroscopy, we examined the structure of the purified Torpedo californica nicotinic acetylcholine receptor in reconstituted dioleoylphosphatidylcholine membranes in H2O and D2O. Using the amide-I band, we calculated the secondary structure of nAChR in H2O to be approx. 19% alpha-helix, 42% beta-structure, 24% turns and 15% unordered. The secondary structure content in D2O was estimated to be 14% alpha-helix, 37% beta-structure, 29% turns and 20% unordered. In the presence of phosphatidic acid the beta-structure content in D2O increased significantly from 37% to 42%. This suggests that an ionic interaction between negatively-charged lipid head groups and positively-charged peptide side chains may stabilize a beta-structure conformation that is necessary for receptor function. The inclusion of cholesterol in the reconstituted membranes significantly increased the alpha-helix content from 14% to 17%. These results support the hypothesis that cholesterol may induce a transmembrane region to undergo a unordered-to-helix transition which is necessary to maintain the integrity of the ion channel. Additionally, we found that nAChR did not undergo major secondary structure changes when subjected to conditions that induce desensitization. This is consistent with the view that the mechanism of desensitization consists of small quaternary rearrangements of the subunits rather than large changes in receptor secondary structure.

Affinity chromatography of neuropathy target esterase.

Neuropathy target esterase (NTE) is a membrane-bound protein which has been proposed as the target site in nerve tissue for initiation of organophosphate induced delayed neuropathy (OPIDN). Efforts to characterize NTE and to determine the mechanism of its involvement in OPIDN have been hampered by the lack of a suitable method for its purification. We describe here the development of a trifluoromethyl ketone liganded affinity gel which selectively binds NTE. Triton X-100/NaCl extracts of NTE from chick embryo brain microsomal membranes were adsorbed to an affinity gel prepared by attachment of 3(9'-mercaptononylthio)-1,1,1-trifluoropropan-2-one to epoxy-activated Sepharose CL4B (MNTFP-Sepharose). Typically 70-80% of NTE activity is bound under conditions in which undetectable quantities of total protein bound (< 4%). It proved difficult to elute active NTE under non-denaturing conditions, but SDS-PAGE analysis of MNTFP-Sepharose bound proteins eluted with 2% SDS identified a 155 kDa NTE-like protein that bound in a trifluoromethylketone- or mipafox-sensitive but paraoxon-insensitive manner. The levels of inhibition of binding correlated with the inhibition of activity and suggested that the 155-kDa band was composed of a single protein. MNTFP-Sepharose affinity chromatography in combination with preparative SDS-PAGE therefore holds promise as a method for obtaining microgram quantities of NTE for chemical analysis and sequencing.

Diterpenoids from Caribbean gorgonians act as noncompetitive inhibitors of the nicotinic acetylcholine receptor.

1. Three cyclic diterpenoids isolated from gorgonians of the Eunicea genus and characterized as eupalmerin acetate (EUAC), 12,13-bisepieupalmerin (BEEP), and eunicin (EUNI) were found to be pharmacologically active on the nicotinic acetylcholine receptor (AChR). 2. The receptor from the BC3H-1 muscle cell line was expressed in Xenopus laevis oocytes and studied with a two-electrode voltage clamp apparatus. 3. All three compounds reversibly inhibited ACh-induced currents, with IC50's from 6 to 35 microM. ACh dose-response curves suggested that his inhibition was noncompetitive. The cembranoids also increased the rate of receptor desensitization. 4. Radioligand-binding studies using AChR-rich membranes from Torpedo electric organ indicated that all three cembranoids inhibited high-affinity [3H]phencyclidine binding, with IC50's of 0.8, 11.6, and 63.8 microM for EUNI, EUAC, and BEEP, respectively. The cembranoids at a 100 microM concentration did not inhibit [alpha-125I]bungarotoxin binding to either membrane-bound or solubilized AChR. 5. It is concluded that these compounds act as noncompetitive inhibitors of peripheral AChR.

The ion channel of muscle and electric organ acetylcholine receptors: differing affinities for noncompetitive inhibitors.

1. Muscle and electric organ acetylcholine receptors (AChR's) were expressed in Xenopus laevis oocytes and differential effects of noncompetitive blockers on each type of receptor were analyzed using a two-electrode voltage clamp. 2. The positively charged channel blockers, phencyclidine (PCP) and tetracaine, displayed a much lower potency on muscle receptor than on the electric organ receptor. The IC50 for both blockers at the electrocyte receptor was close to 1 microM at -60 mV and even lower at more hyperpolarized voltages. In contrast, with muscle receptor IC50's were 20 to 40 microM at -60 or -80 mV. 3. Eupalmerin acetate, an uncharged noncompetitive inhibitor that displaces [3H]PCP from its high-affinity binding site, inhibited both receptors with a similar potency: IC50 of 4.9 and 6.4 microM for electrocyte and muscle receptors, respectively. However, eupalmerin acetate affected the desensitization process in each receptor type differently and triggered an unusual biphasic response in the muscle receptor. 4. These results are discussed with respect to differences in the amino acid sequences of the M2 regions of the two receptors. 5. A third type of noncompetitive inhibitor, Mg2+, was also examined and it inhibited both receptors with a similar potency (IC50, 0.5-1.0 mM). However, Mg2+ appeared to act at sites other than the PCP site.

Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy.

Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.