Tryptophan Scanning Mutagenesis in the αM3 Transmembrane Domain of the Torpedo californica Acetylcholine Receptor:  Functional and Structural Implications

Abstract

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.

Tryptophan-scanning Mutagenesis in the αM3 Transmembrane Domain of the Muscle-type Acetylcholine Receptor

“…In previous studies we have demonstrated that the tryptophan residue, the biggest amino acid, can be inserted along the ␣M3, ␣M4, ␤M3, ␥M3, and ␥M4 lipid-exposed domains of the AChR (25,27,(32)(33)(34)(35). Here, we provide both functional and structural interpretations for the ␣M3 TrpScanM in correlation with previous TrpScanM studies, photolabeling affinity studies (10,12), and the most recent T. marmorata AChR structure (6).…”

“…Functional Interpretation of the ␣M3 Tryptophan Scanning-In contrast to our previous studies (25,27,(32)(33)(34)(35), we have found 6 mutations (␣F280W, ␣V285W, ␣S288W, ␣T292W, ␣V293W, and ␣I294W) that produce significant reductions in the AChR expression levels, suggesting a reduced efficiency of assembly and/or oligomerization of the AChR. Previously, 3-trifluoromethyl-3-(m-[ 125 I]iodophenyl)diazirine) photolabeling studies in the Torpedo californica AChR suggested that these residues are oriented toward the AChR interior, away from the lipid membrane (10).…”

“…The high number of mutations that resulted in non-functional AChRs (ND in Table 1) suggests that the ␣M3 domain is more tightly packed toward the AChR interior than our previous TrpScanM studies (25,32,34,35). According to the cryoelectron microscopic data, the high density of non-functional mutations from ␣Thr-292 to ␣Ile-296 residues is expected due to a highly constricted space among all the AChR transmembrane domains near the cytoplasmic side (6).…”

“…Structural Differences between the Torpedo and Muscle-type ␣M3 Domains-In the study by Guzman et al (32) there are not enough amino acid positions in the C terminus side of the Torpedo AChR ␣M3 domain to allow for a complete comparison with the muscle-type AChR. However, we compared the ␣M3 domains from ␣Leu-279 through ␣Ile-290, which include the N terminus and a large fraction at center of the helix.…”

“…This approach has been used successfully for inward rectifier potassium channels (41)(42)(43), voltage-activated potassium channels (44 -50), nicotinic AChR channels (25,32,34,35), glutamate receptor channels (51), ␥-aminobutyric acid type A receptor channels (52,53), voltage-gated sodium channels (54), N-methyl-D-aspartate receptor channels (55), P2X 4 receptor channels (56), mechanosensitive channels MscL (57), human ) were examined in the current study. MFVAI (blue font) and MLFTMIF*V*IS*S*II (highlighted in turquoise) were studied using tryptophan-scanning mutagenesis and patch clamp (32). V (violet font and italic) and F (blue font and italic) were studied using site-directed mutagenesis and patch clamp (23,31).…”