Complex conformational changes influence and regulate the dynamics of ion channels in living cells. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. We have conducted studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy (PCFM), which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrated PCFM by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer (spFRET) and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of spFRET and self-quenching is widely distributed, which reflects a broad distribution of conformations, including "silent" states of active channels. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer. We have recently applied this new single-molecule experimental approach for studying ion-channel conformational dynamics and mechanisms of NMDA receptors in living cells.
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