Evidence for Ca²⁺‐ and ATP‐sensitive peripheral channels in nuclear pore complexes

Abstract

In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.

Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer

“…Defective nuclear transport in the presence of preserved ATP levels suggests that transport of macromolecules across the nuclear envelope does require maintained nucleotide ratios and a high free energy of ATP hydrolysis (22). Concerted hydrolysis of ATP at the nuclear pore is believed to drive vectorial conformational changes facilitating nuclear transport, with 8-10 molecules of ATP required for a single translocation (40)(41)(42). Moreover, the energy of ATP transformed into the chemical potential of the ran-GTP͞GDP gradient across the nuclear envelope would contribute to the driving force for import and export of macromolecules (2,39).…”

Exceptional mechanical and structural stability of HSV-1 unveiled with fluid atomic force microscopy

“…After lysis, the sample was sonicated in a water bath (three times, 30 seconds each), layered onto a linear 20-45% sucrose gradient (in MNT supplemented with 400 mM NaCl, 1 mM EDTA and 0.5 mM dithiothreitol), and centrifuged in a Beckman SW50.1 rotor at 4000 g for 25 minutes. Capsids were collected as a light-scattering zone from the gradient and resuspended in nuclear isolation medium (NIM) (Shahin et al, 2001) and pelleted by centrifugation for 60 minutes at 4000 g in a Beckman SW28 rotor. The resulting capsid pellet was resuspended in NIM.…”

The nuclear pore complex: structure and function

“…These peripheral channels seem to be absent in yeast NPCs likely due to the smaller size of the central spoke complex (Yang et al, 1998). As of today, the function of these peripheral channels is not ultimately clear and controversially discussed (Hinshaw et al, 1992;Akey, 1995;Soullam and Worman, 1995;Kiseleva et al, 1998;Danker et al, 1999;Shahin et al, 2001;Stoffler et al, 2003;Ohba et al, 2004;Frenkiel-Krispin et al, 2010).…”