Restricted ion flow at the nuclear envelope of cardiac myocytes

Abstract

Flow of small ions across the nuclear envelope (NE) is thought to occur without restriction through large diameter nuclear pore complexes (NPCs). However, investigations with electron and fluorescence microscopy, and with patch-clamp and microelectrode electrophysiology, suggest that in many animal and plant cell types small ions move through a barrier having the signature of large conductance nuclear ion channels (NICs). As nucleocytoplasmic transport and gene activity are regulated by cytoplasmic signals and as it has recently been shown by this investigator that cardiac NICs are sensitive to cAMP-dependent processes (1), it was considered relevant to further investigate the effects of various cytosolic signals on NIC activity. Ion species substitution demonstrated that K+ is the major species responsible for NIC currents. The Na-channel blocker tetrodotoxin (TTX, 100 microM) and the Ca-channel blocker diltiazem (100 microM) had no effect, indicating no relation of NICs to Na- or Ca-channels in transit to the cell surface membrane. Zn2+ (100 microM) blocked NIC activity, suggesting a dual role in nucleocytoplasmic transport and gene function. GTP did not produce measurable effect. However, its nonhydrolyzable analogue GTP-gamma-S (10 microM) suppressed NIC activity, suggesting a role for GTP hydrolysis in NIC function. Deoxynucleotides (dNTPs, 200 microM) produced a transient increase in NIC activity, pointing to a modulation of NIC function by nucleic acid substrates. These results indicate a role for NICs in mediating: (a) control of gene activity by transduction and other cytosolic signals, and (b) nuclear demands and response to such signals.

Calcium, ATP and nuclear pore channel gating

“…NPCC properties from these prostate cancer cells resemble those previously recorded from adult cardiac myocyte nuclei under similar high-K control conditions (e.g., [18,19,35,41]). Channel activity was detected by measuring the time-dependent ion currents across the NE patch, I p =I p (t).…”

“…Nuclei were released by four to six strokes with the loose-fitting pestle of the tissue grinder. Nuclei were purified in Percoll gradient as we previously described for cardiac myocytes [35].…”

“…Their reversal and resting potentials, V rev and V rest , were negligible [19]. To counter potential ion accumulation due to electrical field polarization, voltage pulses were delivered to the pipette electrode from 0 mV, the value at which the current reverses direction (i.e., V rev , [19,35]). Since the outer nuclear membrane of the NE is continuous with the endoplasmic reticulum (ER), a common concern with patch-clamp studies of isolated nuclei is the NE contamination with ER debris (discussed in [18]).…”

“…Single channel conductance, γ, was measured directly from the current jumps (Δi) in each current trace and, indirectly, by multi-Gaussian fitting of the cumulative histograms for each trace ensemble (in this case standard deviation is contaminated by background noise). Gigaseals were obtained without applying suction to the lumen of the recording pipette (i.e., only the capillary suction at the pipette tip was sufficient, see [19,35]). Channel activity was measured with the patch-clamp, nucleus-attached mode, in nuclei devoid of conspicuous ER remains -which could be observed by the 100× (1.36 NA) oil-immersion objective of our inverted microscope [40].…”

Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte

“…With this technique, usually only a few channels per m 2 are observed. These channels frequently had conductances in the range of 0.2 to 0.5 nS (10,11,(19)(20)(21)(22). Only in some cases, single channel conductances of up to 1.7 nS have been observed (9,23).…”