SYNTHETIC PEPTIDES AND 4-HELIX BUNDLE PROTEINS AS MODEL SYSTEMS FOR THE PORE-FORMING STRUCTURE OF CHANNEL PROTEINS .2. TRANSMEMBRANE SEGMENT M2 OF THE BRAIN GLYCINE RECEPTOR IS A PLAUSIBLE CANDIDATE FOR THE PORE-LINING STRUCTURE

A synthetic 23-mer peptide (M2GlyR) with the amino acid sequence of th e putative transmembrane segment M2 of the strychnine-binding alpha su bunit of the inhibitory glycine receptor forms anion-selective channel s in phospholipid bilayers. The most frequent events show single-chann el conductances, gamma, of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes, tau(o), in the millisecond time range. These properties match those of authentic glycine receptors studied in insid e-out patches of cultured rat spinal cord neurons, namely gamma = 27 p S and gamma = 45 pS, and tau(o) in the millisecond time range. The cha nnel activity of M2GlyR is sequence-specific: 1) a synthetic peptide w ith the sequence of putative transmembrane segment M1 (M1GlyR), not co nsidered to contribute to the channel lining, does not form channels; 2) an analog of M2GlyR with site-specific substitutions displays disti nct channel properties: 2 arginine residues at the N and C termini of M2, postulated to contribute to the anion selectivity of the channel, are substituted by glutamic acids, and the analog peptide ([Glu3,22]M2 GlyR) forms cation-selective channels. Further, a four-helix bundle pr otein (T4M2GlyR) formed by tethering four identical M2GlyR modules to a carrier template forms homogeneous anion-selective channels with gam ma = 25 pS in 0.5 M KCl. These channels are blocked by picrotoxin and by the anion channel blockers 9-anthracene carboxylic acid and niflumi c acid, but not by an analog of the local anesthetic lidocaine (QX-222 ), a cation channel blocker. Observed single-channel properties sugges t that a pentameric assembly of alpha and beta subunits with a central pore lined by M2 segments would account for conductance properties of the authentic glycine receptor and the 2 arginines at either end of M 2 could confer anion specificity to the receptor channel.