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

Abstract

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve 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 glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 +/- 0.34 S/cm(2)). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 x 10(6) NPCs. In contrast to earlier 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) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.

Figure 1

Electrical circuit of the nuclear hourglass technique. Two silver wires and a pulse generator are used to drive an alternating current of 1 mA through the capillary. Two conventional KCl-filled microelectrodes are connected to a differential amplifier to measure the voltage drop across the central part of the capillary. The resistance is calculated online from the applied current and measured voltage.

Figure 2

The nucleus inside the capillary and the resulting equivalent circuit. The electrical resistance detected by the nuclear hourglass technique is composed of the resistance of the two NE segments, R_NE1 and R_NE2, and the resistance of the interior of the nucleus, R_Chromatin, in series. The parallel resistor, R_shunt, models the possibility of a substantial amount of the current I bypassing the nucleus through the narrow gap between the inner wall of the capillary and the nucleus. The surface area of the NE segments corresponding to R_NE1 and R_NE2 is calculated by using the geometrical parameters r (the radius of a hypothetical sphere segment) and h (its height) as indicated.

Figure 3

Example of a measurement. The oocyte nucleus is moved into the tapered part of the capillary by a gentle fluid movement. The resulting change in electrical resistance is shown below.

Figure 4

Atomic force microscopy image of a X. laevis oocyte NE spread on mica. The mean density of NPCs is 48 pores/μm².

Figure 5

Independence of NE electrical conductance from active protein transport. The functional state of NPCs is modulated by the addition of cytosolic oocyte extract (transport enabled) and by use of antibodies directed against the nucleoporin gp62 (transport blocked). The NE electrical conductance is virtually unaltered by these maneuvers. Insets show a consensus model of the NPC structure (modified from ref. 34). Although the centrally located transport machinery is in different functional states, the observed conductance may be the result of current flowing through peripheral channels as indicated by arrows.