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.
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 [α-bungarotoxin (α-BTX) and curare] binding, these
results indicate that mAbs could be used
to map functional and structural domains.
The views and conclusions contained In this document are those of the authors and should not be Interpreted as representing the official policies, either expressed or Implied, of the Defense Advanced Research Projects Agency or the U.S. Government.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.