Conductance studies on trichotoxin_A50E and implications for channel structure

Abstract

Trichotoxin_A50E is an 18-residue peptaibol whose crystal structure has recently been determined. In this study, the conductance properties of trichotoxin_A50E have been investigated in neutral planar lipid bilayers. The macroscopic current-voltage curves disclose a moderate voltage-sensitivity and the concentration-dependence suggests the channels are primarily hexameric. Under ion gradients, shifts of the reversal potential indicate that cations are preferentially transported. Trichotoxin displays only one single-channel conductance state in a given experiment, but an ensemble of experiments reveals a distribution of conductance levels. This contrasts with the related peptaibol alamethicin, which produces multiple channel levels in a single experiment, indicative of recruitment of additional monomers into different multimeric-sized channels. Based on these conductance measurements and on the recently available crystal structure of trichotoxin_A50E, which is a shorter and straighter helix than alamethicin, a tightly-packed hexameric model structure has been constructed for the trichotoxin channel. It has molecular dimensions and surface electrostatic potential compatible with the observed conductance properties of the most probable and longer-lived channel.

FIGURE 1

Electron spray ionization-mass spectrum of synthetic trichotoxin_A50E showing the protonated [M+H]⁺ and sodiated [M+Na]⁺ molecular ions at m/z 1689.7 and 1712.1, respectively, and an almost regular series of sequence specific acylium fragment ions b₃-b₁₇ as well as the y₆ fragment ion of the C-terminal hexapeptide at m/z 612.5 (for terminology, see Jaworski and Brückner, 1999). Experimental conditions: Finnigan LCQ instrument; heated capillary temperature 230°C; collision induced energy set at 45%; collision gas, helium; capillary voltage 3 V; spray voltage 4 kV; and direct infusion of 1% peptide in methanol/5% formic acid (1:1, v/v).

FIGURE 2

Macroscopic current-voltage (I-V) curves produced in neutral PC/PE (7:3) planar lipid bilayers bathed by symmetrical 1 M KCl and in the presence of indicated concentrations (cis-side) of trichotoxin_A50E. The peptaibol concentration range was limited both by the membrane dielectric breakdown (near 200 mV) and by a clear intercept with a reference conductance (dashed line, 100 nS).

FIGURE 3

Single-channels induced by 10 nM Trichotoxin (cis-side) in PC/PE (7:3) planar lipid bilayers in symmetrical 1 M KCl. Panel A depicts large conductance channels whereas panel B highlights small conductance channels in different experiments. In all cases, openings are downward deflections. The two upper-most traces on the left of panel A represent a continuous recording of 100 s at an applied voltage of 20 mV. The single-channel current-voltage (I-V) plot on the right-hand side of panel A reveals a quite high conductance of 850–900 pS. At higher voltages, this evolves in “medium-sized” events as shown by the third trace of panel B: an applied voltage of 130 mV induces transitions rapidly flickering between 400 and 200 pS substates. In fewer experiments, a much smaller single-channel conductance is disclosed as shown in panel B. Here, up to six channels of 20 pS each are active in the bilayer.

FIGURE 4

Helical bundle structures for the trichotoxin channel. (Upper left) Hexameric model viewed from the top. (Upper right) Octameric model viewed from the top. (Lower left) Top view of the hexamer showing the electrostatic surface potential. (Lower right) Plot of pore radius versus location along the pore for the hexamer. Figures in the upper row were created using the programs MolScript (Esnouf, 1997) and Raster3D (Merrit and Bacon, 1997).