INTERACTION BETWEEN SON CHANNEL-INACTIVATING PEPTIDES AND ANIONIC PHOSPHOLIPID-VESICLES AS MODES TARGETS

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels hav e concluded that the channel inactivation ''entrance'' (or ''receptor' ' site for the inactivating peptide) consists of a hydrophobic vestibu le within the internal mouth of the channel, separated from the cytopl asm by a region with a negative surface potential. These protein domai ns are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure, We are reporting here on the interaction between the inactivating peptid e of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB- L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation ''entrance,'' contains a hydrophobic domai n (the vesicle bilayer) separated from the aqueous media by a negative ly charged vesicle surface, When challenged by the anionic phospholipi d vesicles, the inactivating ShB peptide 1) binds to the vesicle surfa ce with a relatively high affinity, 2) readily adopts a strongly hydro gen-bonded beta-structure likely an intramolecular beta ''hairpin,'' a nd 3) becomes inserted into the hydrophobic bilayer by its folded N-te rminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptid e 1) binds also to the anionic vesicles, although with a lower affinit y than does the ShB peptide, 2) adopts only occasionally the character istic beta-structure, and 3) has completely lost the ability to traver se the anionic interphase at the vesicle surface and to insert into th e hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate tho se conformed at the inactivation ''entrance'' of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i,e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta- structure, and its insertion into the channel's hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mu tant ShB-L7E peptide to the lack of ability shown by this peptide to c ross through the anionic interphase and insert into the hydrophobic do mains of the model vesicle target.