Effect of avidin on channel kinetics of biotinylated gramicidin.
Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.
Rokitskaya
T I
TI
Belozersky Institute, Moscow State University, Moscow 119899, Russia.
Antonenko
Y N
YN
Kotova
E A
EA
Anastasiadis
A
A
Separovic
F
F
D001360:Avidin / Q000494:pharmacology*; D019921:Biotinylation; D004553:Electric Conductivity; D006096:Gramicidin / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D009942:Organometallic Compounds / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017319:Photosensitizing Agents / Q000378:metabolism
D007211:Indoles; D007473:Ion Channels; D008051:Lipid Bilayers; D009942:Organometallic Compounds; D017319:Photosensitizing Agents; D001360:Avidin; D006096:Gramicidin; C060570:aluminum phthalocyanine