RESUMO
We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Gramicidina/química , Bicamadas Lipídicas/química , Microscopia de Vídeo/métodos , Técnicas de Patch-Clamp/métodos , Carbocianinas , Eletroquímica/métodos , Estudos de Viabilidade , Canais Iônicos/química , Lipossomos/química , Potenciais da Membrana , Membranas Artificiais , Movimento (Física) , Conformação ProteicaRESUMO
Single-molecule spectroscopies in combination with single-channel patch-clamp measurements have the potential for providing new information on ion channel gating processes. Fluorescent gramicidin derivatives could provide a means for calibrating such experiments since the structure of the open channel is known and the mechanism of gating (peptide dimerization) is generally agreed. We describe here the synthesis and characterization of two pairs of gramicidin derivatives that should prove useful for such studies. They contain robust fluorophores, undergo resonance energy transfer (FRET) when they dimerize, and have single-channel properties close to those of the wild-type channel.