N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide

The effects of the inactivating peptide from the eukaryotic Shaker BK + channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr 8 , which exhibit the strongest STD effects; the C4H in the imidazole ring of His 16 ; the methyl protons of Val 4 , Leu 7 , and Leu 10 and the side chain amine protons of one, if not both, the Lys 18 and Lys 19 residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val 4 and Leu 10 , whereas His 16 seems similarly affected as before. Conversely, STD effects on Tyr 8 are strongly diminished, and those on Lys 18 and/or Lys 19 are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of 13 C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a β-hairpin structure when bound to the KcsA channel. Indeed, docking such a β-hairpin structure into an open pore model for K + channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.