Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

Abstract

In many calcium-imaging studies, the nuclear envelope appears to maintain a gradient of free calcium between the nucleus and cytosol. This issue was examined by loading amphibian sympathetic neurons with the calcium indicator fluo 3 via whole-cell patch clamping. Confocal optical sectioning allowed acquisition of independent calibration curves for the nucleus and cytoplasm. Cells were loaded with free calcium levels ranging from 10 nM to 50 microM, using 10 mM BAPTA to control free calcium. The nuclear fluorescence was usually about 130% brighter than the cytoplasmic fluorescence. Had the increased nuclear fluorescence been due to a calcium gradient, then, as fluo 3 was saturated with calcium in both compartments, the fluorescence gradient should have gradually disappeared. Instead, with free-calcium in the pipette set at 50 microM, about five times the level required to nearly saturate fluo 3, the nuclear/cytoplasmic (N/C) fluorescence ratio was not decreased but instead increased slightly. Perfusion of the patch pipette was used in conjunction with imaging to confirm that cytoplasmic fluo 3 was saturated with calcium. After loading cells with 10 nM free calcium, the patch pipette was perfused with high calcium (10 microM). Again, the N/C fluorescence ratio increased at high calcium. The effectiveness of patch-pipette perfusion in changing cellular free calcium levels was indicated by the degree of fluorescence increase--both nuclear and cytosolic compartments showed a roughly 20-fold increase in fluorescence, that is, most of the dynamic range observed in test droplets. To confirm further that cytoplasmic fluo 3 was saturated, cells were perfused with manganese, which binds with very high affinity to fluo 3. Manganese rapidly entered the cytoplasm and nucleus, causing a large increase in fluorescence, but the N/C fluorescence ratio remained relatively constant. Because free manganese in the pipette was 50,000 times the amount required to saturate fluo 3, the greater nuclear fluorescence probably results from additional fluo 3 in the nucleus rather than from calcium or manganese gradients. To gauge further the permeability of the nuclear envelope, the diffusion of calcium was visualized. Under voltage clamp, calcium channels were opened for periods ranging from 5 to 200 msec. Peak calcium levels were observed within 2 microns of the plasma membrane, and declined as calcium diffused into the cell. The nuclear fluorescence increased more than cytosolic fluorescence, but this apparent “amplification” was eliminated by correcting for autofluorescence. Use of cells cultured on glass coverslips and a high- NA microscope objective allowed a satisfactory correction.(ABSTRACT TRUNCATED AT 400 WORDS)

All-or-None Ca2+Release from Intracellular Stores Triggered by Ca2+Influx through Voltage-Gated Ca2+Channels in Rat Sensory Neurons

“…Alternatively, there may be a threshold for luminal Ca 2ϩ (Nelson and Nelson, 1990) mediated by Ca 2ϩ binding proteins within the store interacting with ryanodine receptors (Diaz-Munoz et al, 1990;Gilchrist et al, 1992 Regenerative CICR alters the spatial distribution of the Ca 2؉ signal Ca 2ϩ influx that failed to evoke regenerative CICR produced an elevation in [Ca 2ϩ ] i that was restricted to regions near the plasma membrane (Fig. 6 A,B), similar to previous observations (Hernandez-Cruz et al, 1990;Blumenfeld et al, 1992;O'Malley, 1994). These localized increases in [Ca 2ϩ ] i are in good agreement with a high degree of cytoplasmic Ca 2ϩ buffering in neurons (Miller, 1991).…”

“…6 A,B,F regions (Fig. 6 F), consistent with the idea that Ca 2ϩ freely diff uses through the pores of the nuclear envelope (Al-Mohanna et al, 1994;O'Malley, 1994). A similar spatial distribution was observed when a subthreshold stimulus was applied in the presence of 5 mM caffeine (data not shown).…”

Synaptic Activity Induces Dramatic Changes in the Geometry of the Cell Nucleus: Interplay between Nuclear Structure, Histone H3 Phosphorylation, and Nuclear Calcium Signaling

“…Through a still unresolved mechanism, synaptic activity-induced calcium signals travel to the cell soma where they readily invade the nucleus most likely through NPCs (Nakazawa and Murphy, 1999;Hardingham et al, 2001a;Power and Sah, 2002;Raymond and Redman, 2006;Watanabe et al, 2006;Johenning and Holthoff, 2007;Eder andBading, 2007 Hagenston et al, 2008). In rat hippocampal neurons and amphibian sympathetic neurons, the nuclear compartment border does not sustain a calcium gradient across the nuclear envelope (O'Malley, 1994;Eder and Bading, 2007), although in other cell types the nucleus may be insulated from cytosolic calcium transients (al-Mohanna et al, 1994;Badminton et al, 1996). Mathematical modeling of nuclear calcium dynamics revealed that infoldings divide the nucleus into compartments that can function as nuclear signaling microdomains (Figs.…”

Characterization of a Plasma Membrane Calcium Oscillator in Rat Pituitary Somatotrophs

“…Calcium-mobilizing agonist-induced [Ca 2ϩ ] i oscillations do not occur synchronously within the cell but originate from a specific sub-plasma membrane locus and propagate throughout the cell (41), including the nucleoplasm. The equilibrium between cytoplasmic and nuclear [Ca 2ϩ ] is probably achieved by the release of Ca 2ϩ from nuclear envelope in synchrony with its release from endoplasmic reticulum (42)(43)(44).…”

“…The apparent amplitudes of the Ca 2ϩ signals, expressed as F/F 0 were similar throughout the cytoplasm, including the sub-plasma membrane region, and were higher in the nucleoplasm. These differences should be taken with reservation, because of the controversies in comparing signals originating from different cellular compartments (43)(44)(45). In addition, the lack of difference in the amplitude of [Ca 2ϩ ] i responses close to the plasma membrane and in the central cytoplasmic regions does not rule out the existence of highly localized [Ca 2ϩ ] i elevations in sub-plasma membrane regions, since the resolution of our measurements may not be sufficient to detect such changes.…”