Calcium, ATP and nuclear pore channel gating

Abstract.

Nuclear envelope (NE) cisternal Ca²⁺ and cytosolic ATP are required for nuclear-pore-complex- (NPC-) mediated transport of DNAs, RNAs, transcription factors and other large molecules. Isolated cardiomyocyte nuclei, capable of macromolecular transport (MMT), have intrinsic NPC ion channel behavior. The large ion conductance (γ) activity of the NPC channel (NPCC) is blocked by the NPC monoclonal antibody mAb414, known to block MMT, and is also silenced during periods of MMT. In cardiomyocytes, neither cytosolic Ca²⁺ nor ATP alone directly affects NPCC gating. To test the role of Ca²⁺ and ATP in NPCC activity, we carried out the present patch-clamp study with the pipette attached to the outer NE membrane of nuclei isolated from cultured Dunning G prostate cancer cells. Our investigations demonstrate that in these isolated nuclei neither cytosolic Ca²⁺ nor ATP alone directly affects NPCC gating. However, when simultaneously applied to the bath and pipette, they transiently silence NPCC activity through stimulation of MMT by raising the Ca²⁺ concentration in the NE cisterna ([Ca²⁺]_NE). Our fluorescence microscopy observations with nuclear-targeted macromolecular fluorochromes (B-phycoerythrin and plasmid for the enhanced green fluorescence protein EGFP, pEGFP-C1) and with FITC-labeled RNA support the view that channel silence accompanies MMT. Repeated Ca²⁺ loading of the NE with Ca²⁺ and ATP, after unloading with 1–5 µM inositol 1,4,5-trisphosphate (IP₃), thapsigargin (TSG) or 5 mM BAPTA or EGTA, failed to affect channel gating. This result indicates that other factors are involved in this phenomenon and that they are exhausted during the first cycle of NE Ca²⁺ loading/unloading – in agreement with current theories of NPC-mediated MMT. The results explain how Ca²⁺ and IP₃ waves may convert the NE into an effective Ca²⁺ barrier and, consequently, affect the regulation of gene activity and expression through their feedback on MMT and NPCC gating. Thus, [Ca²⁺]_NE regulation by intracellular messengers is an effective mechanism for synchronizing gene activity and expression to the cellular rhythm.