Tryptophan Substitutions at the Lipid-Exposed Transmembrane Segment M4 ofTorpedo californicaAcetylcholine Receptor Govern Channel Gating

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 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.

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

“…All the positions that were found to increase the macroscopic response after replacement with a tryptophan side chain are lipid-exposed except the N300. Previously we found that the increase in macroscopic response produced by tryptophan replacements at lipid-exposed positions in the alpha M4 domain diminished below the center of the bilayer (Lasalde et al, 1996). In that study a tryptophan replacement at position C412 in the alpha M4 domain did not show any increase in macroscopic response.…”

“…Previous findings demonstrated that a single mutation at the postulated protein-lipid surface of the AChR could produce dramatic effects on the closing transition of the channel (Lee et al, 1994;Lasalde et al, 1996;Ortiz-Miranda et al, 1997;Bouzat et al, 1998;Tamamizu et al, 1999Tamamizu et al, , 2000. These results suggest a possible role of the lipid-exposed regions of the AChR on the gating mechanism.…”

“…The rationale for replacing each of these positions with a tryptophan side chain is based on previous results from our laboratory, which demonstrated that this bulky hydrophobic residue produced significant effects on the AChR channel gating kinetics when located at lipid-exposed regions (Lee et al, 1994;Lasalde et al, 1996;Ortiz-Miranda et al, 1997;Tamamizu et al, 1999Tamamizu et al, , 2000. These seven positions were selected based on their spatial location at the center of the M3 domain of the gamma subunit.…”

The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

“…Mutations along the lipid-facing surface of M4 alter channel gating, suggesting that altered M4-lipid interactions influence coupling between the agonist site and channel gate (21)(22)(23)(24)(25). One model proposes that lipids potentiate gating by enhancing M4 interactions with the adjacent ␣-helices, M1 and M3, to promote effective interactions between the M4 C terminus and the Cys-loop (14), a structure at the interface between the extracellular domain (ECD) and TMD that plays a key role in coupling agonist binding to channel gating (26 -29).…”

Molecular mechanisms of acetylcholine receptor–lipid interactions: from model membranes to human biology

“…Mutations of lipid-exposed residues on M4 alter channel gating showing that there is a direct role for M4 in nAChR function. ( (Li et al 1992;Lee et al 1994;Lasalde et al 1996;Bouzat et al 1998;Tamamizu et al 2000;Mitra et al 2004). As noted, mutation of a lipid facing residue on M4 (αC418W) in the muscle type receptor leads to pathological consequences, demonstrating that M4-lipid interactions are important in Fig.…”