The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053–13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization.
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1 May 2003
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April 28 2003
Tandem Gramicidin Channels Cross-linked by Streptavidin
Tatyana I. Rokitskaya,
Tatyana I. Rokitskaya
Belozersky Institute, Moscow State University, Moscow 119992, Russia
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Elena A. Kotova,
Elena A. Kotova
Belozersky Institute, Moscow State University, Moscow 119992, Russia
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Yuri N. Antonenko
Yuri N. Antonenko
Belozersky Institute, Moscow State University, Moscow 119992, Russia
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Tatyana I. Rokitskaya
Belozersky Institute, Moscow State University, Moscow 119992, Russia
Elena A. Kotova
Belozersky Institute, Moscow State University, Moscow 119992, Russia
Yuri N. Antonenko
Belozersky Institute, Moscow State University, Moscow 119992, Russia
Address correspondence to Yuri Antonenko, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia. Fax: (70) 95-939-31-81; E-mail: [email protected]
*
Abbreviations used in this paper: AlPcS3, aluminum trisulfophthalocyanine; BLM, bilayer lipid membrane; DPhPC, diphytanoylphosphatidylcholine.
Received:
December 30 2002
Revision Received:
March 31 2003
Accepted:
April 02 2003
Online ISSN: 1540-7748
Print ISSN: 0022-1295
The Rockefeller University Press
2003
J Gen Physiol (2003) 121 (5): 463–476.
Article history
Received:
December 30 2002
Revision Received:
March 31 2003
Accepted:
April 02 2003
Citation
Tatyana I. Rokitskaya, Elena A. Kotova, Yuri N. Antonenko; Tandem Gramicidin Channels Cross-linked by Streptavidin . J Gen Physiol 1 May 2003; 121 (5): 463–476. doi: https://doi.org/10.1085/jgp.200208780
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