Topography of nicotinic acetylcholine receptor membrane-embedded domains.
The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in alphaM1, alphaM4, gammaM1, and gammaM4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear alpha-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.
Barrantes
F J
FJ
Instituto de Investigaciones Bioquimicas de Bahia Blanca, B8000FWB Bahia Blanca, Argentina.
Antollini
S S
SS
Blanton
M P
MP
Prieto
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D008081:Liposomes; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014101:Torpedo
D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011978:Receptors, Nicotinic; C024990:asolectin