Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels

Abstract

Melittin produces a voltage-dependent increase in the conductance of planar lipid bilayers. The conductance increases when the side of the membrane to which melittin has been added (cis-side) is made positive. This paper reports observations on the effect of modifying two positively charged amino acid residues within the NH2-terminal region of the molecule: lysine at position 7 (K7), and the NH2-terminal glycine (G1). We have synthesized melittin analogues in which K7 is replaced by asparagine (K7-N), G1 is blocked by a formyl group (G1-f), and in which both modifications of the parent compound were introduced (G1-f, K7-N). The time required to reach peak conductance during a constant voltage pulse was shorter in membranes exposed to the analogues than in membranes modified by melittin. The apparent number of monomers producing a conducting unit for [K7-N]-melittin and [G1-f]-melittin, eight, was found to be greater than the one for [G1-f], K7-N]-melittin and for melittin itself, four. The apparent gating charge per monomer was less for the analogues, 0.5-0.3 than for melittin, one. Essentially similar results were obtained with melittin analogues in which the charge on K7 or G1 or both was blocked by an uncharged N-linked spin label. These results show that the positive charges in the NH2-terminal region of melittin play a major but not exclusive role in the voltage gating of melittin channels in bilayers.

Melittin: a Membrane-active Peptide with Diverse Functions

“…Interestingly, amino-acetylated melittin has also been shown to induce voltage-dependent conductance, indicating that the change in the orientation of melittin under the influence of transmembrane potential is not driven by the interaction of N-terminal or Lys-7 amino groups with the membrane potential (Hanke et al 1983). This is supported by the observation that synthetic melittins with a blocked N-terminal amino group or a Lys-7 to Asn-7 substitution show voltagedependent ion permeability, and the apparent gating charge per monomer was found to be less (0.5-0.3) for these analogues than melittin which has an apparent gating charge of 1 (Tosteson et al 1990). These results show that the positive charges in the amino terminal region of melittin play a major but not exclusive role in the voltage gating of melittin channels in bilayers.…”

“…However, the observation that melittin exhibits multiple conductance levels in POPC (Hanke et al 1983) and asolectin (Tosteson et al 1987) membranes indicates heterogeneity in the 'pore' structure. In addition, as mentioned above, structurally modified analogues of melittin show different concentration dependence on their ion permeability and reduced apparent gating charge per monomer (Tosteson et al 1990). These studies therefore indicate that strictly tetrameric models for the voltage-dependent melittin channel formation are probably not justified (Dempsey 1990).…”

“…The melittin-induced increase in ion permeability is characterized by a fourth power dependence on melittin concentration indicating that a tetrameric structure of melittin may be responsible for channel formation (Tosteson and Tosteson, 1981;Tosteson et al 1990;Stankowski et al 1991). However, the observation that melittin exhibits multiple conductance levels in POPC (Hanke et al 1983) and asolectin (Tosteson et al 1987) membranes indicates heterogeneity in the 'pore' structure.…”

Selective Membrane Permeabilization by the Rotavirus VP5* Protein Is Abrogated by Mutations in an Internal Hydrophobic Domain

“…These residues facilitate cis-trans isomerization of the peptide, permit it to assume a water-soluble conformation, and are suggested to be important for membrane insertion (6). Melittin also forms a voltagegated ion conductance channel in membranes which is size selective and contains a basic domain that presumably permits its association with polar head groups of the membrane (11,59). The influenza M2 protein also forms voltage-gated ion channels in membranes which facilitate virus entry and exit from cells, and M2 pores are blocked by the anti-influenza drug amantadine (26,48,63).…”

AllD-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries

“…The all-D-amino acid enantiomer of melittin was reported to show similar lytic activity towards erythrocyte and bacterial cells as compared to native melittin (Wade et al, 1990). These results suggest that no specific interaction with chiral receptors or enzymes is involved in melittin's mode of action.…”