Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. I. Transmembrane segment M2 of the nicotinic cholinergic receptor channel is a key pore-lining structure

Abstract

Monomeric peptides and four-helix bundle proteins with amino acid sequences of the predicted transmembrane segment M2 of nicotinic acetylcholine receptors (AChR) were designed, synthesized, and used as probes to elucidate the pore-forming structure of the authentic AChR channel. Peptides M2 delta and M2 alpha 4 with sequences of M2 from muscle-like Torpedo californica AChR delta subunit and from rat neuronal AChR alpha 4 subunit form cation-selective channels in lipid bilayers with predominant single-channel conductances in 0.5 M KCl of 20 pS and 27 pS, respectively. Corresponding analogs with presumed pore-lining residues serine 8 or phenylalanine 16 specifically substituted by alanine retain the ability to self-assemble into conductive oligomers and form channels with primary conductances of 16 pS and 22 pS for M2 delta analogs and of 14 pS and 26 pS for M2 alpha 4 analogs. In contrast, peptides with randomized sequences and the same amino acid composition as M2 delta do not form channels. Four-helix bundle proteins T4M2 delta and T4M2 alpha 4 exhibit conductances in 0.5 M KCl of 20 pS and 26 pS. Analogs of T4M2 delta with selective replacement of serine 8 for alanine exhibit lower conductances, whereas substitution of phenylalanine 16 for alanine increases the single-channel conductance. T4M2 delta channels are blocked by open channel blockers such as the quaternary derivative of lidocaine QX-222 and by chlorpromazine. Channel open probability is reduced, and open time is abbreviated. Conduction properties of T4M2 delta and analogs are in accord with several properties of authentic AChRs. These comprehensive studies provide insights into the components of the pore-forming structure of nicotinic AChRs.