Patch clamp detection of transcription factor translocation along the nuclear pore complex channel

Transcription factors (TFs) are cytoplasmic proteins that play an essential role in gene expression. These proteins form multimers and this phenomenon is thought to be one of the mechanisms that regulate transcription. TF molecules reach their DNA binding sites through the large central channel of t...

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Veröffentlicht in:The Journal of membrane biology 1995-08, Vol.146 (3), p.253-261
Hauptverfasser: Bustamante, J O, Oberleithner, H, Hanover, J A, Liepins, A
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container_title The Journal of membrane biology
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creator Bustamante, J O
Oberleithner, H
Hanover, J A
Liepins, A
description Transcription factors (TFs) are cytoplasmic proteins that play an essential role in gene expression. These proteins form multimers and this phenomenon is thought to be one of the mechanisms that regulate transcription. TF molecules reach their DNA binding sites through the large central channel of the nuclear pore complex (NPC). However, the NPC channel is known to restrict the translocation of molecules > or = 20-70 kD. Therefore, during their translocation, TF molecules and/or their multimers may plug the NPC channel and thus, interrupt ion flow through the channel, with a concomitant reduction in the ion conductance of the channel (gamma). Here we show with patch clamp that gamma is reduced during translocation of three major TFs: c-Jun (40 kD), NF-kappa B (approximately equal to 50 kD), and SP1 (approximately equal to 100 kD). Within a minute, femtomolar concentrations of these proteins reduced gamma suggesting a purely mechanical interaction between single TF molecules and the inner wall of the NPC channel. NPCs remained plugged for 0.5-3 hr in the absence of ATP but when ATP was added, channel plugging was shortened to < 5 min. After unplugging, channel closures were rarely observed and the number of functional channels increased. The transcription factors also stabilized the NPCs as shown by the extended duration of the preparations which allowed recordings for up to 72 hr. These observations are the first direct demonstration of the important role of NPCs in mediating nuclear translocation of TFs and, therefore, in forming part of the mechanisms regulating gene expression.
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subjects Adenosine Triphosphate - pharmacology
Animals
Biological Transport - drug effects
DNA - metabolism
Gene Expression Regulation - physiology
Ion Channel Gating
Ion Channels - metabolism
Membrane Potentials - drug effects
Mice
Muscle Proteins - metabolism
Myocardium - metabolism
NF-kappa B - metabolism
Nuclear Envelope - metabolism
Patch-Clamp Techniques
Proto-Oncogene Proteins c-jun - metabolism
Sp1 Transcription Factor - metabolism
title Patch clamp detection of transcription factor translocation along the nuclear pore complex channel
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