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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Bustamante
J O
JO
University of Maryland School of Medicine, Department of Physiology, Baltimore 21201, USA.
Oberleithner
H
H
Hanover
J A
JA
Liepins
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D009124:Muscle Proteins / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism*
D007473:Ion Channels; D009124:Muscle Proteins; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D016329:Sp1 Transcription Factor; D000255:Adenosine Triphosphate; D004247:DNA