INTERACTIONS OF AMINO-TERMINAL DOMAINS OF SHAKER K-CHANNELS WITH A PORE BLOCKING SITE STUDIED WITH SYNTHETIC PEPTIDES

Synthetic peptides of the five alternative NH2-terminal sequences of S haker when applied to the cytoplasmic side of ShB channels that have a n NH2-terminal deletion (ShBDELTA6-46) block the channel with potencie s correlated with the rate of inactivation in the corresponding varian t. These peptides share no sequence similarity and yet three out of th e five have apparent dissociation constants between 2 and 15 muM, sugg esting that the specificity requirements for binding are low. To ident ify the primary structural determinants required for effective block o f ShBDELTA6-46, we examined the effects of substitutions made to the 2 0 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 s howed there was a clear correlation between hydrophobicity and the dis sociation rate constant (k(off)) with little effect on the association rate constant (k(on)). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-term inal half of the peptide, substitutions that increased the net positiv e charge increased k(on) with relatively small changes in k(off), sugg esting the involvement of long-range electrostatic interactions in inc reasing the effective concentration of the peptide. Neutralizing charg ed residues produced small changes in k(off). Charges within the regio n 12-20 act equivalently; alterations which conserved net charge produ ced little effect on either k(on) or k(off). The results are consisten t with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channe l protein and remains relatively unconstrained. Analogous mutations wi thin the NH-2-terminal domain of the intact ShB channel produced quali tatively similar effects on blocking and unblocking rates.