Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.
The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with alpha-[3H]bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor.
Schrattenholz
A
A
Laboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry, 6 Duesbergweg, Johannes-Gutenberg University Medical School, 55099 Mainz, Germany. schratte@mail.uni-mainz.de
Pfeiffer
S
S
Pejovic
V
V
Rudolph
R
R
Godovac-Zimmermann
J
J
Maelicke
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014101:Torpedo
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins