Probing High Permeability of Nuclear Pore Complexes by Scanning Electrochemical Microscopy: Ca²⁺ Effects on Transport Barriers

Abstract

The nuclear pore complex (NPC) solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell to play important biological and biomedical roles. It, however, is not well understood chemically how this biological nanopore selectively and efficiently transports various substances including small molecules, proteins, and RNAs by using transport barriers that are rich in highly disordered repeats of hydrophobic phenylalanine and glycine intermingled with charged amino acids. Herein, we employ scanning electrochemical microscopy to image and measure the high permeability of NPCs to small redox molecules. The effective medium theory demonstrates that the measured permeability is controlled by diffusional translocation of probe molecules through water-filled nanopores without steric or electrostatic hindrance from hydrophobic or charged regions of transport barriers, respectively. The permeability of NPCs, however, is lowered by a low millimolar concentration of Ca 2+ , which can interact with anionic regions of transport barriers to alter their spatial distributions within the nanopore. We employ atomic force microscopy to confirm that transport barriers of NPCs are dominantly recessed (~80%) or entangled (~20%) at the high Ca 2+ level in contrast to authentic populations of entangled (~50%), recessed (~25%), and "plugged" (~25%) conformations at a physiological Ca 2+ level of sub-micromolar. We propose a model for synchronized Ca 2+ effects on the conformation and permeability of NPCs, where transport barriers are viscosified to lower permeability. Significantly, this result supports a hypothesis that the functional structure of transport barriers is maintained not only by their hydrophobic regions, but also by charged regions.

Nanoscale electrostatic gating of molecular transport through nuclear pore complexes as probed by scanning electrochemical microscopy

“…Higher tip currents over each NE patch are attributed to the NPC-mediated transport of protamine from the bottom solution to the tip, which depleted protamine (Fig. 2) to create a local concentration gradient across the NE patch 34. The resultant flux of protamine under the 3 μm-diameter tip was mediated by ∼280 NPCs with a density of 40 NPCs/μm 2 as determined by AFM 34.…”

“…The NE was detached from the nucleoplasm of a large Xenopus oocyte nucleus (∼400 μm in diameter) and spread on the 200 nm-thick microporous region (1.8 mm × 1.8 mm) of a Si 3 N 4 membrane (Fig. 2 and S1†) as established for fluorescence transport studies48 and applied in our recent SECM study 34. The NPCs of micropore-supported NE patches maintain physiological functionalities to mediate the transport of passively impermeable proteins by nuclear transport receptors when the large proteins are labelled with nuclear localization signal peptides 1,49.…”

“…The nucleoplasm-free NE minimized tip fouling to enable SECM imaging and approach curve measurement of multiple NE patches. We employed 10 μm-diameter pores, which were large enough in comparison with a 3 μm-diameter micropipette tip to transport a probe ion under a micropipette tip without hindrance from the pore wall for approach curve measurements 34…”

“…2) to create a local concentration gradient across the NE patch 34. The resultant flux of protamine under the 3 μm-diameter tip was mediated by ∼280 NPCs with a density of 40 NPCs/μm 2 as determined by AFM 34. Pores 1–5 were covered with the NPC-perforated NE, which only partially blocked protamine transport to yield higher tip currents over the centre of each pore than over the surrounding insulating region of the microporous membrane.…”

“…The permeability of NPCs to probe ions is measured by using micropipette-supported liquid/liquid interfaces26,27 as ion-selective tips for scanning electrochemical microscopy28,29 (SECM) and is analysed by effective medium theory30–32 to determine the sizes of the peripheral and central routes. This work is the first to probe polyions at the NPC by SECM in contrast to previous SECM studies of neutral probes or ionic probes with up to three charges at the NPC 15,16,33,34. Significantly, we propose that electrostatic gating, which has been extensively studied for various artificial31,32,35–37 and biological38–41 nanopores, can be relevant to NPCs with much larger pores not only chemically and biologically, but also biomedically to enable efficient nuclear import of therapeutic macromolecules and nanomaterials 4…”