Block of brain sodium channels by peptide mimetics of the isoleucine, phenylalanine, and methionine (IFM) motif from the inactivation gate

Inactivation Of sodium channels is thought to be mediated by an inactivatio n gate formed by the intracellular loop connecting domains III and IV. A hy drophobic motif containing the amino acid sequence isoleucine, phenylalanin e, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these cha nnels, produce two types of block: fast block, which resembles the inactiva tion process, and slow, use-dependent block stimulated by strong depolarizi ng pulses. Fast block by the peptide ac-KIFMK-NH2, measured on sodium chann els whose inactivation was slowed by the ol-scorpion toxin from Leiurus qui nquestriatus (LqTx),was reversed with a time constant of 0.9 ms upon repola rization. In contrast, control and LqTx-modified sodium channels were slowe r to recover from use-dependent block. For fast block, linear peptides of t hree to six amino acid residues containing the IFM motif and two positive c harges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues i n the peptide ac-KIFMK-NH2 with smaller, less hydrophobic residues prevente d fast block. The positively charged tripeptide IFM-NH2 did not cause appre ciable fast block, but the divalent cation IFM-NH(CH2)(2)NH2 was as effecti ve as the pentapeptide ac-KIFMK-NH2. The constrained peptide cyclic KIFMK c ontaining two positive charges did not cause fast block. These results indi cate that the position of the positive charges is unimportant, but flexibil ity or conformation of the IFM-containing peptide is important to allow fas t block. Slow, use-dependent block was observed with IFM-containing peptide s of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH2 showed that large hydrophobic resi dues are preferred in all three positions for slow, use-dependent block. Ho wever, substitution of the large hydrophobic residue diphenylalanine ol the constrained residues phenylglycine or tetrahydroisoquinoline for phe decre ased potency, suggesting that this phe residue must be able to enter a rest ricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM pept ides form two distinct complexes of different stability and structural spec ificity with receptor site(s) on the sodium channel. It is proposed that fa st block represents binding of these peptides to the inactivation gate rece ptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.