INACTIVATING PEPTIDE OF THE SHAKER-B POTASSIUM CHANNEL - CONFORMATIONAL PREFERENCES INFERRED FROM STUDIES ON SIMPLE-MODEL SYSTEMS

Previous studies on the interaction between the inactivating peptide o f the Shaker B K+ channel (ShB peptide, H2N-MAAVAGLYGLGEDRQHRKKQ) and anionic phospholipid vesicles, used as model targets, have shown that the ShB peptide: (i) binds to the vesicle surface with high affinity; (ii) readily adopts a strongly hydrogen-bonded beta-structure; and (ii i) becomes inserted into the hydrophobic bilayer. We now report fluore scence studies showing that the vesicle-inserted ShB peptide is in a m onomeric form and, therefore, the observed;beta-structure must be intr amolecularly hydrogen-bonded to produce a beta-hairpin conformation. A lso, additional freeze-fracture and accessibility-to-trypsin studies, which aimed to estimate how deeply and in which orientation the folded monomeric peptide inserts into the model target, have allowed us to b uild structural models for the target-inserted peptide. In such models , the peptide has been folded near G6 to configure a long P-hairpin mo delled to produce an internal cancellation of net charges in the stret ch comprising amino acids 1-16. As to the positively charged C-termina l portion of the ShB peptide (RKKQ), this has been modelled to be in p arallel with the anionic membrane surface to facilitate electrostatic interactions. Since the negatively charged surface and the hydrophobic domains in the model vesicle target may partly imitate those present at the inactivation 'entrance' in the channel protein [Kukuljan, M., L abarca, P. and Latorre, R. (1995) Am. J. Physiol. Cell Physiol. 268, C 535-C556], we believe that the structural models postulated here for t he vesicle-inserted peptide could help to understand how the ShB pepti de associates with the channel during inactivation and why mutations a t specific sites in the ShB peptide sequence, such as that in the ShB- L7E peptide, result in non-inactivating peptide variants.