Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor

Abstract

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.

Desensitization of neuronal nicotinic receptors

“…Note that even though this new conductance is approximately twofold larger than the normal channel open state, desensitization is still present, consistent with the likelihood of additional closed desensitized states (Revah et al, 1991). Moreover, because of the high affinity of desensitized states for agonists, the mutant ␣7 receptor is now sensitive to both ACh and nicotine in the concentration range that would cause desensitization of wildtype nAChRs (Revah et al, 1991). Interestingly, the desensitized conducting state of the mutant channel can be activated by some competitive antagonists, implying that antagonists, like agonists, can bind to desensitized states with high affinity (Bertrand et al, 1992;Palma et al, 1996;see Sabey et al, 1999 for ␣4␤2 receptors).…”

“…In the above case, these data are best explained by a large decrease in the allosteric constant, L, from 10 5 to 10 1 , also implying for the mutant that approximately 9% of receptors should exist in the D state in the absence of ligand (Corringer et al, 1998). Mutations of a key pore-lining residue, leucine 247 , appear to destabilize the closed conformation of a desensitized state, effectively making it conductive to ions, leading to a dramatic slowing of the time course of desensitization of the usually fast-desensitizing ␣7 receptors (Revah et al, 1991). Note that even though this new conductance is approximately twofold larger than the normal channel open state, desensitization is still present, consistent with the likelihood of additional closed desensitized states (Revah et al, 1991).…”

“…Because homomeric ␣7-nAChRs undergo desensitization, the ␣ subunit must be sufficient to confer this channel property (Courturier et al, 1990;Revah et al, 1991;Seguela et al, 1993). Indeed, this receptor subtype imparts the fastest onset of desensitization (at saturating doses of agonist) of any nAChR, making it (Table 1).…”

“…Mutations of a key pore-lining residue, leucine 247 , appear to destabilize the closed conformation of a desensitized state, effectively making it conductive to ions, leading to a dramatic slowing of the time course of desensitization of the usually fast-desensitizing ␣7 receptors (Revah et al, 1991). Note that even though this new conductance is approximately twofold larger than the normal channel open state, desensitization is still present, consistent with the likelihood of additional closed desensitized states (Revah et al, 1991). Moreover, because of the high affinity of desensitized states for agonists, the mutant ␣7 receptor is now sensitive to both ACh and nicotine in the concentration range that would cause desensitization of wildtype nAChRs (Revah et al, 1991).…”

“…In this respect, the leucine 247 ␣7 subunit mutant exhibits markedly reduced desensitization (Revah et al, 1991;see above), and is lethal when genetically introduced into mice (Orr-Urtreger et al, 1997). Conversely, a polymorphism (serine to phenylalanine) at position 248 in the pore domain of ␣4 subunits results in faster desensitization of ␣4␤2 nAChRs, but causes a form of human cortical epilepsy (Steinlein et al, 1995;Weiland et al, 1996).…”