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.

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.

The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC(50). Taken together, our results suggest that alphaM3 contributes to the gating machinery of the nicotinic ACh receptor and that alphaM3 is comprised of a mixture of two types of helical structures.

The effects of cholesterol on the ion-channel function of the Torpedo acetylcholine receptor (nAChR) and the novel lipid-exposed gain in function ␣C418W mutation have been investigated in Xenopus laevis oocytes. We found conditions to increase the cholesterol/phospholipid (C/P) molar ratio on the plasma membrane of Xenopus oocytes from 0.5 to 0.87, without significant physical damage or change in morphology to the oocytes. In addition, we developed conditions to deplete endogenous cholesterol from oocytes using a methyl-␤-cyclodextrin incubation procedure without causing membrane instability of the cells. Methyl-␤-cyclodextrin was also used to examine the reversibility of the inhibitory effect of cholesterol on AChR function. Depletion of 43% of endogenous cholesterol from oocytes (C/P ‫؍‬ 0.3) did not show any significant change in macroscopic response of the wild type, whereas in the ␣C418W mutant the same cholesterol depletion caused a dramatic gainin-function response of this lipid-exposed mutation in addition to the increased response caused by the mutation itself. Increasing the C/P ratio to 0.87 caused an inhibition of the macroscopic response of the Torpedo wild type of about 52%, whereas the ␣C418W mutation showed an 81% inhibition compared with the responses of control oocytes. The wild type receptor did not recover from this inhibition when the excess cholesterol was depleted to near normal C/P ratios; however, the ␣C418W mutant displayed 63% of the original current, which indicates that the inhibition of this lipid-exposed mutant was significantly reversed. The ability of the ␣C418W mutation to recover from the inhibition caused by cholesterol enrichment suggests that the interaction of cholesterol with this lipid-exposed mutation is significantly different from that of the wild type. The present data demonstrate that a single lipid-exposed position in the AChR could alter the modulatory effect of cholesterol on AChR function.

Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the ␣4␤2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different ␣4␤2 subunit ratios (1␣:4␤, 2␣:3␤, and 4␣:1␤) expressed in Xenopus oocytes. The presence of ␣4 subunit in the oocyte plasmatic membrane increased linearly with the amount of ␣4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the ␣4 subunit. The 2␣4:3␤2 subunit ratio displayed the highest ACh sensitivity. Nicotine doseresponse curves for the 1␣4:4␤2 and 2␣4:3␤2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 M. A biphasic curve for 4␣4:1␤2 was obtained at nicotine concentrations higher than 300 M. The 1␣4:4␤2 subunit ratio exhibited the lowest ACh-and nicotine-induced macroscopic current, whereas 4␣4:1␤2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of ␤2:␣4 subunits increased. All three ␣4␤2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2␣4: 3␤2 subunit ratio. Our data suggest that the subunit ratio of ␣4␤2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate ␣4␤2 receptor activation and desensitization. Neuronal nicotinic acetylcholine receptors (nAChRs)1 belong to a superfamily of ligand-gated ion channels (e.g. ␥-aminobutyric acid, glutamate, 5-hydroxytryptamine, among others) and play an important role in modulating neurotransmitter release in distinct areas of the central and peripheral nervous system (1-5). Nicotine is the active ingredient of tobacco and specifically binds to nAChRs in the brain (3). One of the most remarkable effects of chronic nicotine exposure is the up-regulation of the ␣4␤2 subtype in the central nervous system (6 -10). Another important effect of chronic nicotine exposure is the long lasting functional deactivation of nAChR receptor (11-17). Chronic nicotine exposure produces a loss of nicotinic functional activity as a result of rapid and persistent desensitization (11, 18 -20). Desensitization induced by chronic exposure to nicotine has been hypothesized to trigger the up-regulation of the ␣4␤2 nAChR (3, 6, 21-23). The effect of chronic nicotine exposure on the activity of nAChR subtypes may be related to symptoms associated with nicotine addiction (3, 24, 25) such as tolerance, dependence, and withdrawal. In contrast to the aforementioned studies, a recent work suggests that the ␣4␤2 subtype expressed in the stable cell line K-177 functionally up-reg...