Dibucaine–ion-channel interactions in model systems: A study using fluorescence resonance energy transfer

In 1986, Ondrias et al. [Stud. Biophys. 115 (1986) 17] reported that dibucaine and other local anesthetics reduced the conductivity of membranes containing gramicidin ion channels. They attributed this behavior to a local anesthetic induced swelling of the membrane. Then in 1992, Bridal and Busath [Biochim. Biophys. Acta 1107 (1992) 31] re-examined this system using single channel measurements and found that the channel frequency was reduced, but not the conductance of single channels. They suggested that the mechanism involved a reduction of gramicidin membrane concentration owing to local anesthetic solubilization. In both studies, no direct membrane mediated dibucaine–gramicidin interactions were inferred. It is possible to determine conclusively whether or not direct gramicidin–dibucaine interactions take place using fluorescence resonance energy transfer (FRET). To this end, we have examined the interactions between the ion channel, gramicidin and the antiarrhythmic/local anesthetic, dibucaine, in dioleoylphosphatidylcholine (DOPC) liposomes. FRET was observed for liposomes containing relatively high concentrations of gramicidin and dibucaine, but not for lower concentrations. It can therefore be concluded that there is no specific association between gramicidin and dibucaine in DOPC liposomes. The FRET observed can be attributed to the donors and acceptors situated in close proximity due to their high relative concentration in the liposome. To further examine the nature of the FRET-inducing interactions, the donor (tryptophan) and acceptor (dibucaine) chromophores were examined for FRET in aqueous and lipophilic solutions at sufficiently high concentration to make interactions possible. No interactions were observed for lipophilic solutions, but a quenching of tryptophan fluorescence by dibucaine was recorded in aqueous solution. It is therefore possible that dibucaine can solvate gramicidin in aqueous solution, although we did not observe this to happen for the DOPC liposome solution used in the present study. © 2001 Elsevier Science B.V. All rights reserved.