The following Q&A's have been compiled from questions
received from various scientists since 1991. Some of these have become known as mysteries after a prominent friend of ours introduced the concept. After returning from
the 1998 EMBO Workshop, I decided that it would help to write
down these Q&A's to assist the people trying to understand
our electrophysiological results and interpretations under the
macromolecule-conducting channel paradigm. Most of these ideas
were dealt with in our recent review (Bustamante & Varanda, 1998). Reprints of the paper in HTML or PDF format
can be seen by pressing in the corresponding hiperlinks.
Adobe's PDF Reader can be obtained by pressing here.
YES, it is possible. Only note that you may not be able to measure it if the ion channel activitiy (mean ion flow) is sufficiently high. To understand this concept it suffices to take a look at Ohm's Law (rejected when Georg Ohm first submitted it in 1826) and the basic principles of Electrical Circuit Theory. A high resistance (the gigaseal) in parallel to a low resistance (the nuclear pores) yield an effective, total resistance which can not be greater than the smaller of the resistances. Therefore, one may obtain a gigaseal despite the fact that it may not be measured in some NE patches (i.e., with sufficiently high number of ion-conductingh NPCs). Note also that the gigaseal is revealed during MMT (because the NPCs are plugged during this time) and during the closure of all ion conducting NPCs (because they pose a resistance much greater than that of the gigaseal). If you do not take my word (why should you?), then look at the illustrations of our recordings in all our publications (Bustamante, 1992, 1993, 1994; Bustamante et al., 1995a-c; Bustamante et al., 2000a,b)
NO. First, it has been said that the large conductance ion channel activity recorded from nuclear preparations is not from the NE because during nuclei isolation, there is permanent damaged of the outer NE membrane (ONM) in the form of holes. Our answer to this is that there are no holes in the NE from our isolated nuclei for if this were the case, then no MMT would be possible in our isolated nuclei. Thus, we have shown (Bustamante et al., 1995a, 2000a,b) that both nuclear-targetted B-phycoerythrin (240 kD) and pEGFP (3.1 MD) go into the nucleus when transport substrates are added. Second, it has also been said that since in our nucleus-attached patches the pipette tip is sealed against the ONM, then the large-conductance ion channel activity derives from ion channels of the ONM. Our answer to this is that, even in the worst conditions where no MMT occurs (i.e., pure saline without MMT substrates) small molecules (e.g., FITC-labeled dextrans) pass through the NE, through NPCs (see Clapham's group work). Our experiments confirm this observation and this indicates a virtual short circuit of the bath electrode and the nucleosol (i.e., not the NE cisterna). Therefore, the electrical charge carriers (i.e., the monoatomic ions such as K+) would use the easier pathway between the two electrodes (i.e., the NPC channel). Other interesting hypotheses that we have tested are that the ribosomes are part of the equation (e.g., Simon & Blobel, 1991, 1992). Neither puromycin nor a signal peptide (which modified the ion channel activity associated with the ER) modified the ion channel activity recorded from the NE (see our references).
YES. Isolated nuclei are used in electrophysiology as they are used in other fields (e.g., molecular biology and biochemistry). Interestingly, cell biologists studying nuclear transport avoid such preparation as they know the isolated nuclei may have the nuclear pores artificially closed and inoperative for MMT. For this reason, it is imperative that the investigator always test for the functionality of the NPCs. To this end, we use the methods developed by the experienced nuclear transport people with nuclear-targetted fluorescent macromolecules. For example, we use the highly fluorescent B-phycoerythrin (240 kD) conjugated to the nuclear localization signal (NLS) of the SV40 large T antigen. More recently, we have used the plasmid DNA encoding for the enhanced green fluorescence protein. This work has been published (Bustamante et al., 2000a,b).
NO, the idea of the single NPC having nanotubes surrounding its central big channel was first elaborated by Hinshaw et al. in 1992 on the basis of 3D-reconstruction EM work. Since the publication of this work, reference to the existence of these channels has been made. Our take on this concept is that the ion flow along these nanochannels is never measured as it should be if they connect the cyto- and nucleoplasmic compartments. Therefore, in terms of electrical nucleocytoplasmic contribution, either they do not exist or they are buried inside the NPC in such manner that the main, huge central channel of the NPC shunts any possible flow along them.
YES, this has been published by people working with carbon nanotubes (physicists and physicochemists). These nanotubes, when electrolyte-filled, have a conductance that is voltage-sensitive.
YES, look at the example of the SR Ca2+ channels. They do have a 1,000 pS conductance to K+. Check SR Reviews from more information. Press here for a review.
NO. Plugging is seen in large-conductance mitochondrial ion channels when using peptides targetted to the mitochondrial interior. Press here or here for some refs.
DON'T THEY? Patch-clamp measures directly the ion conductance and demonstrates open-closed channel gating. Fluorescence microscopy (however advanced), goes over several mathematical assumptions, one of which is to assume that the NPCs are always open. This assumptions create a large margin of error which is best explained by the theory of Experimental Physics known as Experimental Error Theory (a.k.a. Error Analysis). A simple explanation of the implications of the errors made through indirect measurements may be found by clicking here. Therefore, the large error inherent to the most advanced fluorescence microscopy approach accounts for the apparent discrepancy.
It may also be of relevance that the most advanced techniques (see Reiner Peters'group), have reported a 5 nm-leak. This leak is much greater than the patch-clamp gigaseal.
DON'T THEY? Patch-clamp measures directly the ion conductance and demonstrates open-closed channel gating. The hour glass technique [see Oberleithner's group - Danker et al., (1999)], places the nucleus inside a tubing. It then assumes that a good seal (something like a gigaseal in patch-clamp) was obtained because in another preparation they measured a good seal.
Please, send me an e-mail message if you think this is right. If you do so, then I will be very happy because I will start quoting you and stop worrying about attaining patch-clamp gigaseals and claim to have one because in 1991 I had one. [Sorry for the exaggeration and cinicism (you can do that in the web, yes?), it is made just to make the point].
The point is that, although the idea of placing the nucleus inside the tubing is brilliant, not until we get the real values of the seal resistance in the same preparation, can we take the data as relevant.
In the 1980s we applied a similar technique to study sodium currents in cardiac myocytes (e.g., Bustamante & McDonald, 1983). This technique is based on that developed at Platon G. Kostyuk's lab for simultaneous voltage-clamp and intracellular perfusion (e.g., Kostyuk et al., 1975; reviewed in Kostyuk, 1984).
In our experience of quite a few years, one can not make the assumption made by Danker et al., (1999) that the seal resistance in one preparation is about the same as that of another.
We look forward to their future experiments (with seal resistance measurements in the same nucleus) validating the conclusions reached in their paper.