Simultaneous measurement of spectroscopic and physiological signals from a planar bilayer system: detecting voltage-dependent movement of a membrane-incorporated peptide.
We developed an experimental system that can measure spectroscopic and physiological signals simultaneously from ion channels in a planar lipid bilayer, to study the relationship between the structure and function of the ion channels. While the membrane potential was clamped, fluorescent emission and ionic currents were measured simultaneously. The fluorescent emissions from a planar bilayer constructed in a specially designed chamber were monitored exclusively, and the signal intensity was measured with a photon-counting system. The intensity of fluorescence and spectral shape were measured successfully from the planar bilayer, with a high signal-to-noise ratio. The system can measure the intensity of fluorescence from a restricted area of the planar bilayer, with a diameter of 70 micrometer and a focal depth of 15 micrometer. The low background signal was achieved by optimizing the optical system. More than 95% of the measured fluorescence comes from the planar lipid bilayer. A 22-mer peptide with a sequence identical to that of the S4 segment of the electric eel sodium channel domain IV was synthesized and fluorescence-labeled. This peptide formed a voltage-dependent ion channel in a planar bilayer. The changes in the intensity of the fluorescence accompanying ionic currents generated by a voltage clamp suggest that voltage gating involves the insertion of the N-terminal of the peptide into the membrane. The electrical and optical signals were measured with a gate time of 10 ms. This measurement enabled the detection of movement of the membrane-incorporated peptides with channel opening.
Hanyu
Y
Y
Biophysics Section, Electrotechnical Laboratory, Ibaraki, Japan. hanyu@etl.go.jp
Yamada
T
T
Matsumoto
G
G
D000595:Amino Acid Sequence; D000818:Animals; D004553:Electric Conductivity; D004593:Electrophorus; D005454:Fluorescence Polarization; D015640:Ion Channel Gating / Q000502:physiology*; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015222:Sodium Channels / Q000378:metabolism / Q000502:physiology; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; D015222:Sodium Channels