Chapter 37 Simultaneous measurement of spectroscopic and physiological signals from a planar bilayer system

Abstract

Publisher Summary This chapter presents an experimental system that can measure spectroscopic and physiological signals simultaneously from ion channels reconstituted in a planar lipid bilayer, to study the relationship between the structure and function of the ion channels. With this system, structural changes of ion channels can be detected with precise spatio-temporal control of their functional state. 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 70μm and a focal depth of 15μm. 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.