Key histidine residues in the nicotinic acetylcholine receptor.
Reactivity of histidine residues of the Discopyge tschudii nicotinic acetylcholine receptor was studied by reaction with DEP and the influence of their modification on functional properties of the receptor was evaluated. Determination of two kinetically distinguishable classes was achieved. The fast-reacting class is composed of 7 histidine residues with an apparent velocity constant k1 = 0.0248 +/- 0.0031 min-1. The second includes--at least--21 histidine residues with a velocity constant k2 = 0.0016 +/- 0.0009 min-1. The circular dichroism spectra of the native receptor and the most DEP-derivative indicate no significant modifications in the alpha-helix content, and fourth derivative spectroscopy analyses show that the environment around the aromatic amino acids remains unchanged. DEP treatment of the receptor results in a time- and reagent concentration-dependent loss of its alpha-bungarotoxin binding ability; these results agree with those obtained with the membrane-bound receptor. The decrease in the neurotoxin binding capacity was correlated with the DEP-reaction extent of the slow groups. Incorporation of 1.93 +/- 0.23 mol of DEP accounted for the maximal binding capacity drop, thus indicating the involvement of two histidine residues per alpha-bungarotoxin binding site. Neither amino groups nor tyrosine residues were modified during the reaction with DEP, indicating that the derivatization of histidine residues is responsible for the observed effect. Faster-reacting residues appear to be involved in agonist-induced ion flux through the nAChR channel. These results strongly support the connection between histidine residues and the receptor functional activity and lead us to infer that the changes observed in alpha-bungarotoxin binding and ionic channel capacity are the consequence of independent events induced by reaction with DEP.
Daniel Lacorazza
H
H
Instituto de Química y Fisicoquímica Biológicas, (UBA-CONICET), Buenos Aires, Argentina.
Otero de Bengtsson
M S
MS
Biscoglio de Jiménez Bonino
M J
MJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate; D004555:Electric Fish; D004557:Electric Organ / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009596:Nitrophenols / Q000378:metabolism; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002038:Bungarotoxins; D007457:Iodine Radioisotopes; D007473:Ion Channels; D008024:Ligands; D009596:Nitrophenols; D011978:Receptors, Nicotinic; D006639:Histidine; D004047:Diethyl Pyrocarbonate