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. However, it 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. However, the permeability of NPCs is reduced by a low millimolar concentration of Ca²⁺, 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 Ca²⁺ level in contrast to authentic populations of entangled (∼50%), recessed (∼25%), and "plugged" (∼25%) conformations at a physiological Ca²⁺ level of submicromolar. We propose a model for synchronized Ca²⁺ 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.

Figure 1.

(A) Scheme of tip-induced transfer of a redox probe molecule (red dots) across the NE supported by a micropore. The tip is positioned over the nucleoplasmic side of the NE. Blue dots represent the product of electrolysis at the tip. The tip–NE distance is given by d. (B) Redox probes used in this study; (ferrocenylmethyl)trimethylammonium, 1,1´-ferrocenedimethanol, hexaammineruthenium(III), and tris(1,10- phenanthroline)cobalt(III).

Figure 2.

Photographs of (A) the swollen nucleus with the NE detached from the nucleoplasm and (B) the nucleoplasmic side of the NE spread on the microporous region of a Si₃N₄ membrane.

Figure 3.

AFM images of (A) cytoplasmic and (B) nucleoplasmic sides of the NE supported by a micropore. (C) AFM image of nuclear baskets as obtained from the nucleoplasmic side of a micropore-supported NE patch.

Figure 4.

Constant-height SECM images of NEs spread over microporous Si₃N₄ membranes in MIB as obtained by using (A) FcTMA⁺ and (B) Ru(NH₃)₆³⁺. Sizes of the respective images are 15 μm × 15 μm and 10 μm × 15 μm. Colored scale bars indicate the normalized tip current. The tip was brought to the left top corner, where the normalized tip current was ~0.70. The tip was scanned laterally at a rate of 1 μm/s with a tip step size of 0.25 μm for both directions.

Figure 5.

(A) SECM approach curves of FcTMA⁺ at micropore-supported NEs prepared in MIB and NIM. Simulated curves at the NE used (a, k) = (0.59 μm, 0.061 cm/s) and (0.45 μm, 0.040 cm/s) for MIB and NIM, respectively, with RG = 2. (B) The corresponding concentration profile of FcTMA⁺ simulated around a nanometer-wide gap between the tip and the NE prepared in MIB. The scale bar indicates the concentration of FcTMA⁺.

Figure 6.

Permeability of micropore-supported NEs plotted against diffusion coefficient for redox probes in MIB and NIM (red and blue symbols, respectively). Permeability values are average values determined from 3–9 approach curves. Solid lines represent the best linear fits.

Figure 7.

(A) AFM image of the cytoplasmic side of a micropore-supported NE prepared in low Ca²⁺ media, MIB. Red, purple, and blue arrows indicate examples of plugged, entangled, and recessed NPCs. (B) Cross sections of the respective NPCs. (C) AFM image of the cytoplasmic side of a micropore-supported NE prepared in high Ca²⁺ media, NIM, where only recessed and entangled NPCs were observed as represented by blue and purple arrows, respectively.

Scheme 1.

Ca²⁺ Effect on Conformational Equilibrium of Transport Barriers of NPCs.