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.

SynopsisSubstance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), a neuromodulator involved in the transmission of pain information, exerts its biological effects by binding to membrane-embedded protein receptors. The influence of membrane lipids on neuropeptide conformation may be critical to these processes. We have characterized in detail the complexes formed between substance P and sodium dodecykulfate (SDS), lysophosphatidylglycerol, and lysophosphatidylcholine micelles. CD spectra of substance P displayed significant induced secondary structure upon addition of these lipids. Potentiometric titration data demonstrated that the pK, of the peptide N-terminal amino group increased from ca. 7.0 to 9.0 in SDS-bound substance P, suggesting direct interaction of the substance P N-terminus with the lipid head-group region. Red shifts in uv spectra of the Phe rings in membrane-bound peptide suggested an increased hydrophobic environment for these substituents. High-resolution one-and two-dimensional correlated spectroscopy nmr spectra displayed differential chemical-shift movements of substance P Gln, Leu, and Met NH protons with added lipid, suggesting involvement of the C-terminal portion of the peptide in the induced secondary structure. The clear influence of the lipid environment on the substance P conformational ensemble suggests a role for the membrane in the events leading to receptor binding.

The interaction of the voltage-dependent channel-forming peptide alamethicin with dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) has been studied using circular dichroism spectroscopy over a range of wavelengths and temperatures. Evidence is presented for the existence of two distinct membrane-bound states of the peptide which reflect different extents of peptide-peptide interaction. An elevated temperature is found to diminish the apparent peptide-peptide interaction. These results provide insight into the general problem of helix-helix interaction in membranes and provide experimental support for the proposal [Popot, J. L., & Engelman, D. M. (1990) Biochemistry 29, 4031-4037] that these interactions can be enthalpically favorable.

The structure of the polypeptide has been determined and a functional model for its mode of action in membranes is presented. Although under some conditions antiamoebin may form ion channels, unlike the closely related alamethicin and zervamicin polypeptides, its major membrane-modifying activity appears to be as an ion carrier.