Electrophoretic plugging of nuclear pores by using the nuclear hourglass technique

The nuclear hourglass technique (NHT) was recently introduced as a novel te chnique that measures the electrical nuclear envelope (NE) conductance of i solated Xenopus laevis oocyte nuclei. The main conclusion drawn from NHT wo rk so far is that nuclear pore complexes (NPCs) of oocytes are in an electr ically open state under physiological conditions, with a mean conductance o f 1.7 nS per NPC. Since nuclear patch-clamp data indicate that usually NPCs are electrically closed, our work has been challenged by the notion that N HT cannot assure a high resistance seal ("gigaseal") between glass wall and NE like that required for patch-clamp experiments. Thus, NHT could have dr amatically underestimated NE electrical resistance. Here we demonstrate tha t NHT does not require a gigaseal for accurate NE conductance measurements. In addition, we present experimental conditions where mean single NPC elec trical conductance is reduced 26-fold due to electrophoretic plugging by ne gatively charged nucleoplasmic macromolecules. In addition, data indicate t hat under physiological conditions (i.e., when macromolecules are offered i n the cytosolic solution) the nuclear surface is heavily folded, underestim ating "true" NE surface by a factor of 2.6. When "true" NE surface area is taken into consideration, modified values of mean single NPC conductances o f 654 pS for electrically open conditions and 25 pS for electrically plugge d conditions can be calculated. We conclude that the large overall NE conductance detected with the nuclear hourglass technique in intact Xenopus laevis oocyte nuclei can be explaine d by the sum of single NPC conductances in the pS range, as long as open pr obability is high. This confirms previous patch-clamp work concerning singl e NPC conductance, but disagrees with the view that mean open probability o f NPC channels is usually low.