SYNTHETIC PEPTIDES AND 4-HELIX BUNDLE PROTEINS AS MODEL SYSTEMS FOR THE PORE-FORMING STRUCTURE OF CHANNEL PROTEINS .1. TRANSMEMBRANE SEGMENT M2 OF THE NICOTINIC CHOLINERGIC RECEPTOR-CHANNEL IS A KEY PORE-LINING STRUCTURE

Monomeric peptides and four-helix bundle proteins with amino acid sequ ences of the predicted transmembrane segment M2 of nicotinic acetylcho line receptors (AChR) were designed, synthesized, and used as probes t o elucidate the pore-forming structure of the authentic AChR channel. Peptides M2delta and M2alpha4 with sequences of M2 from muscle-like To rpedo californica AChR delta subunit and from rat neuronal AChR alpha4 subunit form cation-selective channels in lipid bilayers with predomi nant single-channel conductances in 0.5 M KCl of 20 pS and 27 pS, resp ectively. Corresponding analogs with presumed pore-lining residues ser ine 8 or phenylalanine 16 specifically substituted by alanine retain t he ability to self-assemble into conductive oligomers and form channel s with primary conductances of 16 pS and 22 pS for M2delta analogs and of 14 pS and 26 pS for M2alpha4 analogs. In contrast, peptides with r andomized sequences and the same amino acid composition as M2delta do not form channels. Four-helix bundle proteins T4M2delta and T4M2alpha4 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 in creases the single-channel conductance. T4M2delta channels are blocked by open channel blockers such as the quaternary derivative of lidocai ne QX-222 and by chlorpromazine. Channel open probability is reduced, and open time is abbreviated. Conduction properties of T4M2delta and a nalogs are in accord with several properties of authentic AChRs. These comprehensive studies provide insights into the components of the por e-forming structure of nicotinic AChRs.