PATCH-CLAMP DETECTION OF TRANSCRIPTION FACTOR TRANSLOCATION ALONG THENUCLEAR-PORE COMPLEX CHANNEL

Transcription factors (TFs) are cytoplasmic proteins that play an esse ntial role in gene expression. These proteins form multimers and this phenomenon is thought to be one of the mechanisms that regulate transc ription. TF molecules reach their DNA binding sites through the large central channel of the nuclear pore complex (NPC). However, the NPC ch annel is known to restrict the translocation of molecules greater than or equal to 20-70 kD. Therefore, during their translocation, TF molec ules and/or their multimers may plug the NPC channel and thus, interru pt ion flow through the channel, with a concomitant reduction in the i on conductance of the channel (gamma). Here we show with patch clamp t hat gamma is reduced during translocation of three major TFs: c-Jun (4 0 kD), NF-kappa B ( approximate to 50 kD), and SP1 ( approximate to 10 0 kD). Within a minute, femtomolar concentrations of these proteins re duced gamma suggesting a purely mechanical interaction between single TF molecules and the inner wall of the NPC channel. NPCs remained plug ged 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 ex tended duration of the preparations which allowed recordings for up to 72 hr. These observations are the first direct demonstration of the i mportant role of NPCs in mediating nuclear translocation of TFs and, t herefore, in forming part of the mechanisms regulating gene expression . The studies also demonstrate the potential of the patch clamp techni que in quantifying TF translocation to the nucleus, mRNA export, and o ther processes governing gene expression.