NH2-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

A synthetic, channel-forming peptide, derived from the alpha -subunit of th e glycine receptor (M2GlyR), has been synthesized and modified by adding fo ur lysine residues to the NH2 terminus (N-K-4-M2GlyR). In Ussing chamber ex periments, apical N-K-4-M2GlyR (250 muM) increased transepithelial short-ci rcuit current (I-sc)by 7.7 +/- 1.7 and 10.6 +/- 0.9 mA/cm(2) in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are sign ificantly greater than those previously reported for the same peptide modif ied by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwam oto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K-4-M2GlyR caused a concentration-dependent inc rease in I-sc (k([1/2]) = 190 muM) that was potentiated two- to threefold b y 1-ethyl-2-benzimidazolinone. N-K-4-M2GlyR-mediated increases in I-sc were insensitive to changes in apical cation species. Pharmacological inhibitor s of endogenous Cl- conductances [glibenclamide, diphenylamine-2-dicarboxyl ic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2 ,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little e ffect on N-K-4-M2GlyR-mediated I-sc. Whole cell membrane patch voltage-clam p studies revealed an N-K-4-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activa tion. Planar lipid bilayer studies yielded results indicating that N-K-4-M2 GlyR forms a 50-pS anion conductance with a k([1/2]) for Cl- of 290 meq. Th ese results indicate that N-K-4-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.