A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor

Abstract

The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single-channel conductances of systematically mutatedTorpe& receptors expressed in Xenoprts oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholinc receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From Ihc results obtained. we conclude that a ring of uncharged polar residues comprising threonine 244 of the a-subunit (a-l-244), pS250. yT253 and 6S258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed a-helical configuration ofthe Mt-containing transmembranc segment. together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the y-subunit. are asymmetrically positioned at the channel constriction.Nicotinic acetylcholine receptor; Ionic channel: Channel constriction: Site-directed mutagenesis: cDNA expression: Single-channel recording INTRSDUCTIONThe nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein composed of 4 kinds of homologous subunits which are assembled in the molar stoichiometry a&G and arranged pseudosymmetritally around a central cation-conducting channel (see [I] for review). It is generally assumed that the hydrophobic segments Ml-M4 of the 5 subunits ofthe AChR form membrane-spanning a-helices and that polar side chains of some of the transmembrane segments line the channel pore [2]. By analysing single-channel current properties of site-specific mutants of the AChR. we have previously identified 3 clusters of negatively charged and glutamine residues as major determinants of the rate of ion transport through the AChR channel [3]. These amino acid clusters, neighbouring the M2 segments of the constituent subunits of the AChR. are suggested to form three (cytoplasmic. intermediate and extracellular) anionic rings (see Fig. IA The present investigation deals with further characterization of the ion-conducting pore of the AChR channel. We systematically mutated uncharged polar amino acid residues of the Torpedo AChR subunits positioned between the cytoplasmic ring and the extracellular ring to alter the size and polarity of their side chains. Some other mutations were also introduced into the y-subunit to alter its unique sequence features. Mutant AChR channels were expressed in Xenoplrs oocytes by microinjection of mRNAs derived from specifically mutated cDNAs and were analysed for single-channel conductance. The results obtained indicate that a cluster of uncharged polar residues. which probably forms a ringlike structure adjacent to the intermediate ring in the assumed a-helical configuration of the MZ-containing transmembrane segment, is critical for determining the rate of ion transport. The present investigation. in conjunction with our previous studies. suggests that this uncharged polar rin...

The functional architecture of the acetylcholine nicotinic receptor explored by affinity labelling and site-directed mutagenesis

“…The star denotes the wild type residue in this subunit. Mutations in the a-, /?-and y-subunits yield similar results (see Imoto et al 1991) to those of the 5-subunit.…”

“…Mutations in this ring indeed affect the conductance for Rb + and Cs + ions Imoto et al 1991), yet, without changing the permeability preference for the physiological ions K + > Na + > Li + . Further studies are thus needed to understand the mechanism of ion selectivity, in particular the structure which governs anion versus cation selectivity.…”

“…Mutation into Val of residues from the chlorpromazine labelled Threonine ring (Villarroel et al 1991 for rat receptor; Imoto et al 1991 for T. californica receptor), caused a slight decrease (about 20 %) of the conductance for K + , whereas its replacement by Ala or Gly slightly increased it (by about 10%).…”

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

“…Charge inversion substitutions at the extracellular 20Ј and intracellular Ϫ4Ј rings of the T. californica ␣-subunit resulted in a 50% reduction in ␥. Introduction of the bulky Tyr residue in place of the smaller Thr or Ser at the 2Ј central ring position also reduced ␥ by 50% (14), suggesting that the pore narrows considerably in this region. Recent data indicate the most constricted region of the adult muscle nACh receptor to be at positions Ϫ1Ј and Ϫ2Ј (13).…”

Structure of the Transmembrane Cysteine Residues in Phospholamban

“…In a fashion similar to that of tryptophan in gramicidin, mutation of T244 in the acetylcholine receptor has dramatic effects on ion conductance. Mutation of T244 can either enhance or restrict the flow of ions through the ion channel depending on the volume of the substituted residue (Imoto et al, 1991;Villarroel et al, 1991).…”