Structural rearrangements in single ion channels detected optically in living cells.

Abstract

Total internal reflection fluorescence microscopy was used to detect single fluorescently labeled voltage-gated Shaker K(+) channels in the plasma membrane of living cells. Tetramethylrhodamine (TMR) attached to specific amino acid positions in the voltage-sensing S4 segment changed fluorescence intensity in response to the voltage-driven protein motions of the channel. The voltage dependence of the fluorescence of single TMRs was similar to that seen in macroscopic epi-illumination microscopy, but the exclusion of nonchannel fluorescence revealed that the dimming of TMR upon voltage sensor rearrangement was much larger than previously thought, and is due to an extreme, approximately 20-fold suppression of the elementary fluorescence. The total internal reflection voltage-clamp method reveals protein motions that do not directly open or close the ion channel and which have therefore not been detected before at the single-channel level. The method should be applicable to a wide assortment of membrane-associated proteins and should make it possible to relate the structural rearrangements of single proteins to simultaneously measured physiological cell-signaling events.