T. Danker et al., Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte, P NAS US, 96(23), 1999, pp. 13530-13535
Citations number
33
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.