Aqueous Solubilization of Transmembrane Peptide Sequences with Retention of Membrane Insertion and Function

Abstract

We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (I(SC)) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.

Channel Replacement Therapy for Cystic Fibrosis

“…To decrease these deficiencies, the M2GlyR sequence was modified systematically by adding up to six lysyl or diaminopropionic acid (DAP) residues to either the C-or N-terminus (Tomich et al, 1998). This study examined a number of physical, pharmacological and physiological characteristics of the adducted sequences.…”

Effect of the Synthetic NC-1059 Peptide on Diffusion of Riboflavin across an Intact Corneal Epithelium

“…44 The peptide was designed so that the transmembrane segment had an N-terminal tetra-lysine domain and so that a central leucine was flanked by a palindrome that is based on the Nterminal portion, and was designed so that the transmembrane segment had an N-terminal tetra-lysine domain and with a central leucine defining a palindrome based on the N-terminal portion of second transmembrane segment of the alpha subunit of the spinal cord glycine receptor M2GlyR and was designated NC-1059 (KKKKAARVGL-GITTVLVTTIGLGVRAA). 45 The peptides were purified and characterized by reversed-phase HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy.…”

Synthetic, Biologically Active Amphiphilic Peptides

“…Tomich and co-workers [26] [45] have modified the 23-residue sequence of the M2 segment of the glycine-gated Cl -channel. The sequence is H 2 N-PARVG-LGITT-VLTMT-TQSSG-SRA-COOH.…”