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...

Two series of two-phase anaerobic systems, consisting of a hyperthermophilic (80°C) reactor and a thermophilic (55°C) reactor, fed with a mixture of kitchen garbage (KG) and polylactide (PLA), was compared with a single-phase thermophilic reactor for the overall performance. The result indicated that ammonia addition under hyperthermophilic condition promoted the transformation of PLA particles to lactic acid. The systems with hyperthermophilic treatment had advantages on PLA transformation and methane conversion ratio to the control system. Under the organic loading rate (OLR) of 10.3 g COD/(L day), the PLA transformation ratios of the two-phase systems were 82.0% and 85.2%, respectively, higher than that of the control system (63.5%). The methane conversion ratios of the two-phase systems were 82.9% and 80.8%, respectively, higher than 70.1% of the control system. The microbial community analysis indicated that hyperthermophilic treatment is easily installed to traditional thermophilic anaerobic digestion plants without inoculation of special bacteria.

To gain an insight into the molecular basis of ion permeation mechanism through the nicotinic acetylcholine receptor (AChR) channel, we have determined permeability ratios of organic cations relative to Na+ of specifically mutated Torpedo californica AChR channels expressed in Xenopus oocytes. The mutations involved mainly the side chains of the amino acid residues in the intermediate ring, where mutations have been found to exert strong effects on single-channel conductance and ion selectivity among alkali metal cations. The results obtained reveal that both the size and the net charge of the side chains of the intermediate ring are involved in determining the permeability, and provide experimental evidence that the pore size at the intermediate ring is a critical determinant of permeability. Our findings further suggest that changes in net charge exert effects on permeability by affecting the pore size of the channel.

Interactive comment on "Chemical de-staining and the delta correction for blue intensity measurements of stained lake subfossil trees" by Feng Wang et al. Feng Wang et al.

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