Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor.
Nicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T5M2 delta, that emulates the presumed pore-forming structure of this receptor was generated by assembling five helix-forming peptide modules at the lysine epsilon-amino groups of the 11-residue template [K*AK*KK*PGK*EK*G], where * indicates attachment sites. Helical modules represent the sequence of the M2 segment of the Torpedo californica acetylcholine receptor (AChR) delta subunit; M2 segments are considered involved in pore-lining. Purified T5M2 delta migrates in SDS-PAGE with an apparent M(r) approximately 14,000, concordant with a protein of 126 residues. T5M2 delta forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M KCl is 40 pS. This value approximates the 45 pS single channel conductance characteristic of authentic purified Torpedo AChR, recorded under otherwise identical conditions. These results, together with conformational energy calculations, support the notion that a bundle of five amphipathic alpha-helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel.
Montal
M O
MO
Department of Biology, University of California, San Diego, La Jolla 92093-0319.
Iwamoto
T
T
Tomich
J M
JM
Montal
M
M
D000595:Amino Acid Sequence; D003198:Computer Simulation; D004553:Electric Conductivity; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000502:physiology
D007473:Ion Channels; D011506:Proteins; D011978:Receptors, Nicotinic