Voltage-dependent translocation of R18 and DiI across lipid bilayers leads to fluorescence changes.

We show that the lipophilic, cationic fluorescent dyes R18 and Dil translocate from one monolayer of a phospholipid bilayer membrane to the other in a concentration and voltage-dependent manner. When the probes were incorporated into voltage-clamped planar membranes and potentials were applied, displacement currents resulted. The charged probes sensed a large fraction of the applied field. When these probes were added to only one monolayer, displacement currents were symmetrical around 0 mV, indicating that the probes distributed equally between the two monolayers. Charge translocation required that the bilayer be fluid. When membranes were in a condensed gel phase, displacement currents were not observed; raising the temperature to above the gel-liquid crystalline transition restored the currents. Translocation of R18 was also shown by fluorescence measurements. When R18 was in the bilayer at high, self-quenching concentrations, voltage pulses led to voltage-dependent fluorescence changes. The kinetics of the fluorescence changes and charge translocations correlated. Adding the quencher I- to one aqueous phase caused fluorescence to decrease or increase when voltage moved R18 toward or away from the quencher at low, nonquenching concentrations of R18. In contrast to R18, Dil incorporated into bilayers was a carrier fo I-, and hence I- altered Dil currents. Voltage-driven translocations allow R18 and Dil to be used to probe membrane potential changes.

Structure-function of the channel-forming colicins.

The channel-forming colicins are plasmid-encoded bacteriocins that kill E. coli and related cells and whose mode of action is of interest in related problems of protein import and toxicology. Colicins parasitize metabolite receptors in the outer membrane and translocate across the periplasm with the aid of the Tol or Ton protein systems. X-ray structure data for the channel domain and colicin are available. Residues have been identified that affect the channel ion selectivity and particular helices implicated in channel structure and in conformational changes required for binding or insertion of the channel into the membrane. Unique aspects of the colicin channel system are the involvement of protein import in the gating process, the existence of multiple open and closed states, and the existence and action of an immunity protein that involves specific intramembrane helix-helix interactions with transmembrane helices of the colicin channel-forming domains.