Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte

Nuclear pore complexes (NPCs) mediate both active transport and passive dif fusion across the nuclear envelope (NE). Determination of NE electrical con ductance. however, has been confounded by the lack of an appropriate techni cal approach. The nuclear patch clamp technique is restricted to preparatio ns with electrically closed NPCs, and microelectrode techniques fail to res olve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glas s tube, which narrows in its middle part to 2/3 of the diameter of the nucl eus. The isolated nucleus is sucked into the narrow part of the capillary b y gentle fluid movement, while the resulting change in electrical resistanc e is monitored. NE electrical conductance was unexpectedly large (7.9 +/- 0 .34 S/cm(2)), Evaluation of NPC density by atomic force microscopy showed t hat this conductance corresponded to 3.7 x 10(6) NPCs. In contrast to earli er conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically "open" state with a mean single NPC electrical conductance of 1.7 +/- 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) reveale d this large NPC conductance to be independent of the activation state of t he transport machinery located in the center of NPCs. We conclude that peri pheral channels, which are presumed to reside in the NPC subunits, establis h a high ionic permeability that is virtually independent of the active pro tein transport mechanism.