IL1-receptor accessory protein-like 1 (IL1RAPL1), a protein involved in cognitive functions, regulates N-type Ca2+-channel and neurite elongation

Abstract

Null mutations in the IL1-receptor accessory protein-like 1 gene (IL1RAPL1) are responsible for an inherited X-linked form of cognitive impairment. IL1RAPL1 protein physically interacts with neuronal calcium sensor-1 (NCS-1), but the functional impact of the IL1RAPL1/NCS-1 interaction remains unknown. Here, we demonstrate that stable expression of IL1RAPL1 in PC12 cells induces a specific silencing of N-type voltage-gated calcium channels (N-VGCC) activity that explains a secretion deficit observed in these IL1RAPL1 cells. Importantly, this modulation of VGCC activity is mediated by NCS-1. Indeed, a specific loss-of-function of N-VGCC was observed in PC12 cells overexpressing NCS-1, and a total recovery of N-VGCC activity was obtained by a down-regulation of NCS-1 in IL1RAPL1 cells. The functional relevance of the interaction between IL1RAPL1 and NCS-1 was also suggested by the reduction of neurite elongation observed in nerve growth factor (NGF)-treated IL1RAPL1 cells, a phenotype rescued by NCS-1 inactivation. Because both proteins are highly expressed in neurons, these results suggest that IL1RAPL1-related mental retardation could result from a disruption of N-VGCC and/or NCS-1-dependent synaptic and neuronal activities.

Fig. 1.

HVA Ca²⁺ currents of reduced amplitude at IL1RAPL1-expressing PC12 cells. (A) Whole-cell Ca²⁺ current in naïve cells. Typical Ca²⁺ currents (lower traces) recorded in PC12 cells and I/V curve are presented. Note the presence of LVA and HVA currents. (Scale bars: 50 pA and 25 msec.) (B₁) Whole-cell average Ca²⁺-current densities in SHAM (black line) and IL1RAPL1 (blue lines) PC12 cells. (Top) Western blot denoting the presence of IL1RAPL1 protein (≈83-kDa) in stably transfected PC12 cells (arrow) but not in SHAM cells. Ca²⁺ current traces induced by depolarization (Middle) from −90 mV to +70 mV by 15-mV, 30-ms steps (Bottom Right). (Scale bars: 1 pA/pF and 5 msec.) (B₂) The corresponding I/V curves. Note the reduced amplitude of the HVA (at +35 mV, Left Lower) but not LVA (at −10 mV, Left Upper) current densities in IL1RAPL1 cells (blue traces). ∗, P < 0.05; ∗∗∗, P < 0.001. (Scale bars: 2 pA/pF and 8 msec.)

Fig. 2.

Expression of IL1RAPL1 in PC12 cells abolishes N-type Ca²⁺ current. (A) Same presentation as in Fig. 1B₁. Average traces obtained at +35 mV (Inset) from naïve, SHAM, and IL1RAPL1 cells are shown. Cell numbers are indicated above the traces. Black traces indicate Ca²⁺ currents under control conditions; red traces indicate Ca²⁺ currents in the presence of 1 μM ω-conotoxin GVIA. (Scale bars: 2 pA/pF and 8 msec.) (B) Mean plateau-current amplitude (20- to 30-msec time-window) recorded at +35 mV in the same cells as in A. ∗∗∗, P < 0.001. ns, not significant. The N-type contribution to the HVA current (gray bars) was obtained by subtracting GVIA currents from those in control condition.

Fig. 3.

N-type current is depressed in PC12 overexpressing NCS-1. (A) PC12 cells were transfected with plasmids allowing expression of GFP or YFP (YFP/GFP) or expression of YFP-tagged NCS-1 (NCS-1/YFP) or NCS-1 siRNA and GFP (siNCS-1+GFP). (B₁) Data presentation is identical as that in Fig. 2A. Cells submitted to recording were identified by epifluorescence. (Scale bars: 2 pA/pF and 8 msec.) (B₂) Presentation is the same as that in Fig. 2B.

Fig. 4.

NCS-1 is needed for inhibition of N-type current by IL1RAPL1 in PC12 cells. (A) IL1RAPL1 PC12 cells transfected with plasmids allowing expression of GFP alone (+GFP) or NCS-1 siRNA with GFP (siNCS-1+GFP). Data presentation is similar to that in Fig. 2A. Recorded cells were identified by epifluorescence. (Scale bars: 2 pA/pF and 8 msec.) (B) Same presentation as in Fig. 2B. ∗, P < 0.05.

Fig. 5.

NGF induces neurite extension and increase in N-type Ca²⁺ current in PC12 cells. (A) Näive/SHAM cells plus NGF. (Left) Phase-contrast photograph of neuronal-like differentiated PC12 cells after NGF treatment. (Scale bar: 20 μm.) (Right) Distribution histogram of neurites' length emitted by naïve/SHAM cells in absence (ctrol, white bar) or presence of NGF for 8 days (gray bar). (Inset) Mean neurite length. ∗∗∗, P < 0.001. (B) Ca²⁺ currents are presented as in Fig. 2. For distinction between “no-diff” and “diff” cells, see Results. NGF-differentiated PC12 cells (diff) have a cell capacitance >13 pF and visible neurites. (Scale bars: 2 pA/pF and 8 msec.) ∗∗, P < 0.005.

Fig. 6.

IL1RAPL1 inhibits NGF-induced neurite elongation and N-type current. (A) IL1RAPL1 cells plus NGF. The presentation is the same as in Fig. 5A. (Scale bar: 20 μm.) (B) Same presentation as in Fig. 5B. (Scale bars: 2 pA/pF and 8 msec.)

Fig. 7.

IL1RAPL1 inhibits NGF-induced neurite elongation and N-type current by way of NCS-1. (A) IL1RAPL1 cells plus NGF. The presentation is the same as in Fig. 6A. Note the morphological difference between the GFP-expressing cells (bright cell) and the surrounding nontransfected IL1RAPL1 cells. (B) (Left) Same presentation as in Fig. 2A. (Scale bars: 2 pA/pF and 8 msec.) (Right) The corresponding I/V curves. Note the increased amplitude of the HVA, but not LVA currents, in “diff” cells (dark green traces). ∗, P < 0.05.