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

Abstract

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 (γM3). 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 voltageclamped 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 αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC 50 s were similar to those measured for the αC418W 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.

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).…”

Nicotinic Receptor M3 Transmembrane Domain: Position 8′ Contributes to Channel Gating

“…The four transmembrane domains are likely to be involved in the conformational changes associated with gating. According to few lines of experimental evidence showing the contribution of M3 to channel gating [Campos-Caro et al (1997), Wang et al (1999), Cruz-Martin et al (2001)], the present study demonstrates that the M3 segments of all muscle subunits influence both opening and closing steps and reveals the mechanistic and structural bases underlying the contribution to function of position 8Ј.…”

“…A few lines of experimental evidence indicate that M3 is a key component of the nAChR channel gating apparatus: 1) by using ␣7/␣3 chimeric subunits, Campos-Caro et al (1997) demonstrated that the M3 domain influences the gating of neuronal nAChRs; 2) a mutation at position 9Ј of M3 of the ␣ subunit has been found in a patient suffering from a congenital myasthenic syndrome (Wang et al, 1999). Kinetic analysis of engineered mutant nAChRs revealed that this position is essential for the opening and closing of the nAChR (Wang et al, 1999); and 3) tryptophan substitutions at lipid-exposed residues of the ␥ M3 transmembrane domain of T. californica nAChR increased the macroscopic currents of the resulting nAChRs (Cruz-Martin et al, 2001).…”

Structure and Function of the Glycine Receptor and Related Nicotinicoid Receptors

“…As described above, movements of M3 residues in the nicotinicoid receptors have been inferred by differential accessibility in SCAM studies. Similarly, systematic tryptophan mutagenesis in M3 of the nAChR subunits has shown that movement of M3 contributes to the gating machinery of these receptors (54). The periodicity of the observed effects suggest a mixture of helical structures in M3 (55).…”