Calcium, ATP and nuclear pore channel gating

Nuclear envelope (NE) cisternal Ca2+ and cytosolic ATP are required for nuc lear-pore-complex (NPC-) mediated transport of DNAs, RNAs, transcription fa ctors and other large molecules. Isolated cardiomyocyte nuclei, capable of macromolecular transport (MMT), have intrinsic NPC ion channel behavior. Th e large ion conductance (gamma) activity of the NPC channel (NPCC) is block ed by the NPC monoclonal antibody mAb414, known to block MMT, and is also s ilenced during periods of MMT. In cardiomyocytes, neither cytosolic Ca2+ no r ATP alone directly affects NPCC gating. To test the role of Ca2+ and ATP in NPCC activity, we carried our the present patch-clamp study with the pip ette attached to the outer NE membrane of nuclei isolated from cultured Dun ning 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 micro scopy observations with nuclear-targeted macromolecular fluorochromes (B-ph ycoerythrin and plasmid for the enhanced green fluorescence protein EGFP, p EGFP-C1) and with FITC-labeled RNA support the view that channel silence ac companies MMT. Repeated Ca2+ loading of the NE with Ca2+ and ATP, after unl oading with 1-5 mu M inositol 1,4,5-trisphosphate (IP3), thapsigargin (TSG) or 5 mM BAPTA or EGTA, failed to affect channel gating. This result indica tes that other factors are involved in this phenomenon and that they are ex hausted during the first cycle of NE Ca2+ loading/unloading - in agreement with current theories of NPC-mediated MMT. The results explain how Ca2+ and IF, waves may convert the NE into an effective Ca2+ barrier and, consequen tly, affect the regulation of gene activity and expression through their fe edback on MMT and NPCC gating. Thus, [Ca2+](NE) regulation by intracellular messengers is an effective mechanism for synchronizing gene activity and e xpression to the cellular rhythm.