Calcineurin involvement in the regulation of high-threshold Ca2+ channels in NG108-15 (rodent neuroblastoma x glioma hybrid) cells

Abstract

1. We examined the relationship between calcineurin (protein phosphatase 2B (PP2B) and voltage-operated Ca2+ channels (VOCCs) in NG108-15 cells. PP2B expression in NG108-15 cells was altered by transfection with plasmid constructs containing a full length cDNA of human PP2B beta(3) in sense (CN-15) and antisense (CN-21) orientation. 2. Confocal immunocytochemical localization showed that in wild-type cells, PP2B immunoreactivity is uniformly distributed in undifferentiated cells and located at the inner surface of soma membrane and neurites in differentiated cells. 3. To test the Ca2+ dependence of the VOCC, we used high-frequency stimulation (HFS). The L- and N-type VOCCs decreased by 37 and 52%, respectively, whereas the T-type current was only marginally sensitive to this procedure. FK-506 (2 microM), a specific blocker of PP2B, reduced the inhibition of L- and N-type VOCCs induced by HFS by 30 and 33%, respectively. 4. In CN-15-transfected cells overexpressing PP2B, total high-voltage-activated (HVA) VOCCs were suppressed by about 60% at a test potential of +20 mV. Intracellular addition of EGTA or FK-506 into CN-15-transfected cells induced an up to 5-fold increase of HVA VOCCs. 5. These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca(2+)-dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect on N-type VOCCs.

Figure 3. Effects of high-frequency stimulation (HFS) on HVA VOCCs

I-V recordings of pharmacologically separated L-type VOCCs with 1 μM ωCgTX GVIA in the presence of 0.1 mM Ni²⁺ in bath solution (A and C) and N-type VOCCs with 10 μM nifedipine (B and D) were obtained in control conditions (curves 1 and 4), immediately after HFS (curves 2 and 5) and 5 min after HFS (curves 3 and 6) in two different cells. Bath solution contained 0.1 mM Ni²⁺. Inset shows the HFS protocol for the experiment, where V_t is test potential corresponding to I-V_max.

Figure 1. Current-voltage relationships of inward Ca²⁺ currents in NG108-15 cells

Currents were recorded in control conditions, without any blockers. Recordings were made from differentiated cells without (A) or with (B) neurites after 5 days of differentiation in control conditions. The dotted line in B shows the same I-V curve presented in A for comparison.

Figure 2. High-voltage-activated VOCCs in differentiated NG108-15 cells

Recordings were made in Ca²⁺-Ni²⁺ containing bath solution (10 mM Ca²⁺, 0.1 mM Ni²⁺). Current responses on stepping from the holding potential (V_h = −80 mV) to different test potentials (A and C) are shown with corresponding I-V relationships (B and D) evoked by sequential 5 or 10 mV depolarizing steps from the holding potential (V_h = −80 mV (▪ in B, • in D) and −50 mV (▴)). The two HVA components, L-type (A and B) and N-type (C and D) were pharmacologically separated by 1 μM ωCgTX GVIA (A and B) or 10 μM nifedipine (C and D), respectively.

Figure 4. Similarity in the effects of ωCgTX GVIA and HFS on VOCCc composing T-, N- and residual types

I-V recordings of VOCCs were made in two different cells in solution containing 10 μM nifedipine before (curves 1 and 4) and after 1 μM ωCgTX GVIA application in Ca²⁺-free application solution (A, curve 2) or HFS (B, curve 5), and in the presence of 0.1 mM Ni²⁺ at V_h = −50 mV (curves 3 and 6).

Figure 5. Influences of FK-506 on HFS effects in differentiated NG108-15 cells

I-V recordings of pharmacologically separated L-type VOCCs by 1 μM ωCgTX GVIA (A) and N-type VOCCs by 10 μM nifedipine (B) were obtained in control conditions (curves 1 and 3) and immediately after HFS (curves 2 and 4) in cells which were patched with a pipette solution containing 2 μM FK-506. Amplitude histograms (means ±s.d.) for integral of the I-V relationships (Σ_Ca) in FK-506-containing solution (C and D) and similar histograms obtained in control conditions (E and F) are presented. Bath solutions contained 0.1 mM Ni²⁺.

Figure 6. Immunoblot analysis of PP2B expression in mock, sense and antisense PP2B transfectants

Undifferentiated (Undiff) and differentiated (Diff) NG108-15 cells (10 μg protein) were treated as described in the Methods. Anti-PP2B IgG was used at a dilution of 1/1000, as with immunocytochemical experiments. A major band of about 58 kDa was detected (arrow), corresponding to the shorter of two mammalian PP2B isoenzymes PP2B-A2. Mock-transfected cells (wild-type (WT)) were compared with cells transfected with sense (CN-15) or antisense (CN-21) constructs of PP2Bβ₃ (HT6/6) cDNA. Higher molecular weight bands correspond to tightly bound PP2B-A and PP2B-B oligomers that are recalcitrant to dissociation under the solubilization conditions used.

Figure 7. Intracellular localization of PP2B immunoreactivity in NG108-15 cells

PP2B distribution in non-differentiated NG108-15 cells. Cells were incubated with anti-PP2B IgG (1/1000) and visualized by confocal immunofluorescence microscopy. Phase images of wild-type (A), sense (B) and antisense (C) transfectants are presented in the upper panels. Corresponding immunofluorescence images (lower panels) represent merged images. D, immunostaining with anti-PP2B IgG (1/1000) in permeabilized sense transfectants. Control experiments show the lack of staining in non-permeabilized transfectants (E) or in transfectants with immunostaining by pre-immune serum (1/1000) (F). Corresponding phase images are presented above. Each image represents ten merged 2 μm optical slices.

Figure 8. PP2B distribution and VOCC activity in differentiated NG108-15 cells

Cells were incubated with anti-PP2B IgG (1/1000) and visualized by confocal immunofluorescence microscopy. Phase images of wild-type (A), sense (B) and antisense (C) transfectants are presented in the upper panels and fluorescence images are represented in the middle panels. Corresponding I-V relationships of VOCCs were measured in control solutions without blockers (bottom panels). Each point represents the mean ±s.d. Each image represents ten merged 2 μm optical slices. Inset shows a fluorescence image of a single 2 μm optical slice through the cell middle (A).

Figure 9. Sensitivity of different Ca²⁺ current components to PP2B overexpression in differentiated NG108-15 cells

The mean ±s.d. peak amplitude of Ca²⁺ current evoked by test potentials from V_h = −80 mV to −20, −15, 0 and +20 mV corresponding to the peak of I-V curves of T-, Q-, L- and N-type channels for given experimental conditions (10 mM Ca²⁺in bath solution) without blockers are presented for wild-type (□), sense (▪)- and antisense ()-transfected cells. Each histogram value represents the values calculated from data presented in Fig. 8. *P < 0·0002 with respect to control.

Figure 10. Expression of Ca²⁺ channels in differentiated sense NG108-15 transfectants (CN-15)

A, leak-subtracted membrane current recorded in control bath solutions in CN-15 transfectants in response to voltage ramps from holding potential of −90 mV to peak potential of +90 mV during 0.5 s steps in control conditions (a ramp rate of 0.36 V s⁻¹). B, ramps recorded in the presence of 10 mM EGTA in pipette solution from CN-15 cells. C, ramp recordings obtained in the presence of 35 μM FK-506 from CN-15 cells. Numbers adjacent to the curves indicate the sequence of trace recordings with respect to time. Insets show the current traces normalized to their peaks that were induced by 500 ms pulses of depolarization corresponding to the peak (+20 mV) of the I-V curve.

Figure 11. Recovery of VOCC inhibition induced by HFS of transfected (CN-15) cells during suppression of PP2B activity

Leak-subtracted membrane current recorded in response to application of voltage ramps from holding potential of −90 mV to peak potential of +90 mV during 0.5 s steps in control condtions (A), in the presence of 10 mM EGTA (B) and 35 μM FK-506 (C) in the pipette solution are shown for transfected CN-15 cells, in control bath solution without any blockers. Numbers near the curves indicate sequence of trace recording in time and traces recorded immediately after HFS marked by asterisks with the exception of trace 3 in A, which corresponds to traces obtained at the same time after patching as traces 12 in B and C.