Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds

Abstract

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds.

Antimicrobial peptides in toroidal and cylindrical pores

“…Later studies reported that mutations of Q7 perturbed the channel stability [32, 34]. In the simulations of the alamethicin tetramer in a toroidal pore and of the alamethicin hexamer in a cylindrical pore, we observed that the pores dried up.…”

Voltage-dependent energetics of alamethicin monomers in the membrane

“…Lateral association of the peptides is enhanced by the interhelical interaction between Gln7, Glu18, Gln19 (hydrophilic convex surface). The importance of Gln7 inter-helix hydrogen bonds is evident from mutational studies where Q7A substitution was found to abolish channel formation [45]. Here again the peptides do not cross the bilayer completely.…”

Intrinsic rectification of ion flux in alamethicin channels: studies with an alamethicin dimer

“…The bend in the helix axis around Pro14 leads to a funnel-shaped pore with a wider diameter at the C-terminal end than at the N-terminus. The narrowest region is at the level of the Gln7 residues, which may form an intermolecular ring of hydrogen bonds (Molle et al, 1996).…”