Patch-clamp detection of macromolecular translocation along nuclear pores

Abstract

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus.

Dendrimer-assisted patch-clamp sizing of nuclear pores

“…On the basis of the reported NPC geometry and electrical conductivity of the medium, the predicted ion conductance (γ) for a single NPC channel (NPCC) is ≅10 2 -10 3 pS (e.g., [4]). The NPCC passive diameter changes (e.g., 20-70 kDa, 3-12 nm) with cell type and the phase of the cell cycle (e.g., [2]).…”

“…The cargo must contain a nuclear localization signal (NLS), a nuclear export signal (NES), or both, and its translocation is dependent upon a gradient of Ran-GDP/GTP, ATP, cargo receptors (importins or exportins that form the translocating complex), etc. (e.g., [2,3,4,5]). …”

“…However, patch-clamp investigations indicate that the NPC behaves like an ion channel (e.g., [6,7], see [4]), with gates opening and closing in a probabilistic manner (e.g., [8]). Patch-clamp demonstrates that, despite the large NPCC diameter, the NPC gate(s) is(are) sufficient to regulate ion flow into and out of the nucleus through changes in its open probability (see [4]).…”

“…However, patch-clamp investigations indicate that the NPC behaves like an ion channel (e.g., [6,7], see [4]), with gates opening and closing in a probabilistic manner (e.g., [8]). Patch-clamp demonstrates that, despite the large NPCC diameter, the NPC gate(s) is(are) sufficient to regulate ion flow into and out of the nucleus through changes in its open probability (see [4]). Open-close channel gating as well as channel plugging (e.g., [6,9,10,11]) explain why it is possible to observe significant ion gradients across the nuclear envelope (NE) and thus nuclear ion regulation (e.g., [12]).…”

“…A logical continuation of our patch-clamp studies (e.g., [6,8,19,20,21]) would be to attempt to incorporate the EM method of pore sizing with direct measurements of the NPCC electrical conductance, γ, using patch-clamp. As previously explained [10], the central idea of using particles of known diameter to size a membrane channel with electrical methods is similar to the resistive pulse principle of Coulter counters and other technologies.…”