The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle

Abstract

NCC27 is a nuclear chloride ion channel, identified in the PMA-activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. Electrophysiological studies in Chinese hamster ovary (CHO-K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. We also demonstrate that Cl- ion channel blockers known to block NCC27 led to arrest of CHO-K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.

Figure 1. Northern blots of whole tissues probed with NCC27 cDNA

A, human fetal tissue: (1) brain, (2) lung, (3) liver, (4) kidney; human adult tissue: (5) heart, (6) brain, (7) placenta, (8) lung, (9) liver, (10) skeletal muscle, (11) kidney, (12) pancreas (13) spleen, (14) thymus, (15) prostate, (16) testis, (17) ovary, (18) small intestine, (19) colon, (20) peripheral blood leucocyte. B, whole mouse embryos: (1) day 7 (2) day 11 (3) day 15 (4) day 17; mouse tissue: (5) heart, (6) brain, (7) spleen, (8) lung, (9) liver, (10) skeletal muscle, (11) kidney, (12) testis.

Figure 2. Comparison of channel properties from NCC27-transfected CHO-K1 cells and untransfected CHO-K1 cells

The upper panels are single-channel current traces recorded at different membrane potentials from NCC27-transfected (left) and untransfected (right) CHO-K1 cells. The lower panels show current-voltage (i–V) relationships (left) and channel open times (right), derived from 500 ms of data at each potential (▴, transfected cells; ▪, untransfected cells). The biophysical properties of the channel obtained from the two cell types appear to be identical (see text).

Figure 3. CHO-K1 cells expressing a histone-GFP fusion protein

Photograph of CHO-K1 cells expressing a histone-GFP fusion protein as viewed under fluorescence microscopy (A), phase contrast microscopy (B) and an overlay of images from A and B (C).

Figure 4

A and B, whole-cell patch-clamp recordings of NCC27-transfected CHO-K1 cells from either round (A) or flat (B) cells. Current amplitudes were measured at 350 ms from the voltage step onset. The current amplitude was 60 % greater in round cells (A) than in flat cells (B). The i–V relationships differ between the cell types (top right panel: ▪, round cells; •, flat cells). C, inside-out experiments using macropatch configuration (top) and single-channel recording (bottom), both done using similar transmembrane chloride concentrations to those present in the whole-cell studies. The lower i–V curve plots the averages obtained from all four such patches in which only a single channel was present. The single-channel i–V relationships are very similar to those in the whole-cell recordings from round cells (above), consistent with the hypothesis that NCC27 is responsible for the current increment seen in round cells.

Figure 6. Effect of anti-FLAG m2 monoclonal antibody on the whole-cell current of amino terminal FLAG-transfected NCC27 cells (n = 5)

The two upper left panels show currents from the same cell before (top) and 7 min after (bottom) exposure to m2 antibody. The i–V curves on the top right show combined results for 5 such experiments (▪, control; •, after exposure to m2 antibody). Both inward and particularly outward currents are markedly reduced by m2 antibody, without a shift in the reversal potential. The bottom left traces are the m2 antibody-sensitive component of current (derived by subtracting the two records above). The i–V curve for this antibody-sensitive current (lower right) closely resembles that recorded from untransfected round CHO-K1 cells (Fig. 5).

Figure 5. Comparison of whole-cell currents recorded from round, i.e. dividing (n = 8; left), and flat, i.e. non-dividing (n = 8; right), untransfected CHO-K1 cells

The flat cells have markedly less current than the round cells on average, although 4-5 % of flat cells exhibit similar currents to those seen in all round cells (see inset on the right). The similarity of the i–V plots (middle left and inset on the right) also suggest the presence of the same channel. Bottom panels show micrographs of round (left) and flat (right) cells as viewed under a phase contrast microscope.

Figure 7. Effects of chloride ion channel blockers on single-channel NCC27 currents

The top panel demonstrates the effect of IAA-94. Current blockade begins within 30 s and is complete at 3 min, but recovers fully and rapidly. A9C (middle panel) produces rapid, complete but irreversible block. DIDS had no effect on the NCC27 single-channel current (bottom panel).

Figure 8. Histograms showing relative distribution of DNA content (x-axis) from CHO-K1 cells (cell number, y-axis)

Untreated control (A), DMSO control (B), 1 mM DIDS (C) and 1 mM IAA-94 (D).

Figure 9. Line graphs from 3 independent experiments demonstrating the effects of increasing concentrations of Cl⁻ channel blockers on the proportion of CHO-K1 cells in G2/M phase (expressed as % change from control)

DIDS had no effect at any concentration (left). A9C significantly increased the fraction in G2/M at 2 mM (P < 0.0003), but not at 1 mM. IAA-94 produced a dose-dependent increase at all concentrations studied (P < 0.0001), with the effect appearing to plateau above 1 mM.