Gaegurin 4, a peptide antibiotic of frog skin, forms voltage‐dependent channels in planar lipid bilayers

Abstract

Gaegurin 4 (GGN4) is a cationic peptide of 37 amino acids (MW 3748) isolated from the skin of Rana rugosa. It has shown a broad spectrum antimicrobial activity in vitro against Gram-negative and -positive bacteria, fungi and protozoa. To understand its mechanism of antimicrobial action, we examined the effect of GGN4 on the membrane conductance and the electrical properties of GGN4-induced pores in planar lipid bilayers under voltage clamp. Natural and synthetic GGN4 (0.01-1 microg/mL) increased the membrane conductance in a concentration-dependent manner, but GGN4 (1-23), an N-terminal fragment of the peptide with little antimicrobial activity, failed to increase the conductance. At symmetrical 100 mM KCI, unitary conductances of about 120 pS were frequently observed. Their current-voltage relations were linear and open state probabilities were close to 1, but longer closing events were seen more frequently at negative voltages. In addition, GGN4-induced pores were selective for cation over anion, the permeability ratio of K+ to Cl- being 6: 1 in neutral and 7: 1 in acidic lipid bilayers. In conclusion, our results indicate that GGN4 forms voltage-dependent and cation-selective pores in planar lipid bilayers. The ionophoric property of GGN4 is likely to contribute to its antimicrobial activity.

Mechanisms of Selective Antimicrobial Activity of Gaegurin 4

“…As reported previously (Kim et al, 1999), GGN4 (0.01∼ 1 μg/ml) induced membrane currents immediately after applying it to planar lipid membranes. Fig.…”

“…Planar lipid bilayers (Kim et al, 1999) were formed by painting lipid solution over an aperture (200 μM) of a plastic cup (0.6 ml) using a fire-polished glass rod in a buffer containing 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) adjusted to 7.4 with 1 M N-methyl d-glucamine (NMDG) or KOH. Lipid solutions were made by a mixture of phospholipids, cholesterol, or extracted lipids at a certain proportion of each lipid in n-decane (25 mg/ml).…”

“…Several lines of evidences indicate that the antimicrobial activity of GGN4 results from its pore-forming activity. For example, GGN4 forms cation-selective and voltage-dependent channels in lipid membranes (Kim et al, 1999). Estimation of diameters of GGN4-induced pores by analytical ultracentrifugation indicates that GGN4-induced pores are composed of 5 GGN4 molecules or more with a diameter of 7.3 Å (Eun et al, 2006).…”

Structure and Function of Membrane-Lytic Peptides

“…Alternatively, within a neutral membrane surface anions accumulate in the proximity of the positively charged peptides. Channel activities have also been tested for protegrins (Sokolov et al, 1999), gaegurin 4 (Kim et al, 1999), cecropin-melittin hybrids, indolicidins, bacteneccins, polymyxin B , catfish cathelicidin (Basanez et al, 2002) and many other peptides (Kourie and Shorthouse, 2000).…”

Fuscopeptins, antimicrobial lipodepsipeptides from Pseudomonas fuscovaginae, are channel forming peptides active on biological and model membranes

“…This was indeed the case for other related LDPs so far analyzed, like SRE [23][24][25][26]32] and SP25A [22]. Nevertheless, cationic selectivity seems to be a common characteristic of positively charged channel forming peptides, like mastoparan and mast cell degranulation peptide [33,34], the antimicrobial cathelicidin tritrpticin [35], gaegurin from frog skin [36], synthetic basic α-helical peptides [37], a segment of the sodium channel polypeptide [38] (see also the excellent review of Sansom [33], and that more recent of Kourie [39]).…”