Permeability of Single Nuclear Pores

Abstract

In this first application of optical single transporter recording (OSTR), a recently established technique for optically monitoring the activity of single transporters in membrane patches (Tschödrich-Rotter and Peters. 1998. J. Microsc. 192:114-125), the passive permeability of the nuclear pore complex (NPC) was measured for a homologous series of hydrophilic probe molecules. Nuclei were isolated from Xenopus oocytes and firmly attached to filters containing small cylindrical pores. Transport through membrane patches spanning filter pores was measured by scanning microphotolysis. Thus the permeability coefficients of single NPCs were determined for fluorescently labeled dextrans of approximately 4, 10, and 20 kDa. Dextrans of >/=40 kDa could not permeate the NPC. The data were consistent with a model in which the NPC contains a single diffusion channel. By application of established theories for the restricted diffusion through small pores, the diffusion channel was approximated as a cylinder with a radius of 4.4-6.1 nm (mean 5. 35 nm). Because the transport rate constant of the single NPC was known, the equivalent length of the channel could be also determined and was found to be 40-50 nm (mean 44.5 nm). The symmetry of the NPC implies that a singular component such as the diffusion channel is located at the center of the NPC. Therefore a common transport pathway apparently mediates both passive and signal-dependent transport. To test this hypothesis, measurements of signal-dependent transport and of the mutual effects signal-dependent and passive transport may exert on each other are in progress.

Calcium, ATP and nuclear pore channel gating

“…Furthermore, small dextran molecules (e.g., <10 kDa) placed outside the nucleus translocate into the nuclear interior and not into the NE cisterna [13,14,15], demonstrating that only the NPCCs, and not channels at either of the two NE membranes, provide an electrical and chemical shortcircuit between the pipette and bath electrodes in nucleus-attached patch-clamp preparations. Recent independent patch-clamp and fluorescence microscopy studies confirm this view [16,17]. That is, nuclei isolated at our laboratory (e.g., [18,19]) and elsewhere (e.g., [15]) show that the NE cisterna is isolated from the extra-and intra-nuclear compartments.…”

From the trap to the basket: getting to the bottom of the nuclear pore complex

“…Although the directionality of transport would be driven predominantly by the RanGTP gradient across the two sides of the NPC to create a continuous bidirectional cascade of cargo Richards et al 1997), we concur that after being captured, the process by which cargo complexes negotiate the high density FG-barrier and are funneled into the central pore through weak and transient, rather than strong interactions, is as yet unknown. Nevertheless, the application of single molecule optical approaches by Peters and coworkers to study the kinetics of molecular transport through NPCs may provide further insight into such effects (Keminer and Peters 1999;Radtke et al 2001;Kiskin et al 2003;Hoppener et al 2005;Kubitscheck et al 2005).…”

How to operate a nuclear pore complex by Kap-centric control

“…Despite bearing a 50 nm-diameter central channel, only molecules smaller than 40 kDa or 5 nm in size can passively diffuse in an unhindered manner through the NPC, whereas larger macromolecules are excluded. 3 In contrast, rapid transport is accorded to nuclear transport receptors such as karyopherins (Kaps, also known as importins and exportins) that identify, bind and shuttle large specific cargoes that would otherwise be rejected by the NPC in the absence of Kaps. 4 Kaps distinguish essential cargoes such as transcription factors from other non-essential proteins based on peptide sequences known as nuclear localization or nuclear export signals (NLS or NES).…”