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. 2013 Mar 1;126(Pt 5):1199-206.
doi: 10.1242/jcs.117135. Epub 2013 Jan 23.

CALHM1 controls the Ca²⁺-dependent MEK, ERK, RSK and MSK signaling cascade in neurons

Ute Dreses-Werringloer  1 , Valérie Vingtdeux, Haitian Zhao, Pallavi Chandakkar, Peter Davies, Philippe Marambaud
Affiliations

CALHM1 controls the Ca²⁺-dependent MEK, ERK, RSK and MSK signaling cascade in neurons

Ute Dreses-Werringloer et al. J Cell Sci. .

Abstract

Calcium homeostasis modulator 1 (CALHM1) is a Ca(2+) channel controlling neuronal excitability and potentially involved in the pathogenesis of Alzheimer's disease (AD). Although strong evidence indicates that CALHM1 is required for neuronal electrical activity, its role in intracellular Ca(2+) signaling remains unknown. In the present study, we show that in hippocampal HT-22 cells, CALHM1 expression led to a robust and relatively selective activation of the Ca(2+)-sensing kinases ERK1/2. CALHM1 also triggered activation of MEK1/2, the upstream ERK1/2-activating kinases, and of RSK1/2/3 and MSK1, two downstream effectors of ERK1/2 signaling. CALHM1-mediated activation of ERK1/2 signaling was controlled by the small GTPase Ras. Pharmacological inhibition of CALHM1 permeability using Ruthenium Red, Zn(2+), and Gd(3+), or expression of the CALHM1 N140A and W114A mutants, which are deficient in mediating Ca(2+) influx, prevented the effect of CALHM1 on the MEK, ERK, RSK and MSK signaling cascade, demonstrating that CALHM1 controlled this pathway via its channel properties. Importantly, expression of CALHM1 bearing the natural P86L polymorphism, which leads to a partial loss of CALHM1 function and is associated with an earlier age at onset in AD patients, showed reduced activation of ERK1/2, RSK1/2/3, and MSK1. In line with these results obtained in transfected cells, primary cerebral neurons isolated from Calhm1 knockout mice showed significant impairments in the activation of MEK, ERK, RSK and MSK signaling. The present study identifies a previously uncharacterized mechanism of control of Ca(2+)-dependent ERK1/2 signaling in neurons, and further establishes CALHM1 as a critical ion channel for neuronal signaling and function.

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Figures

Fig. 1.
Fig. 1.
Effect of CALHM1 expression on kinase activation. (A,B) HT-22 cells transiently transfected with CALHM1 or empty vector were challenged with Ca2+ add-back conditions for 20 min (see the Materials and methods section). Cell lysates were probed on phospho-protein arrays (A). Densitometric analysis was performed, and results are expressed as fold change compared with empty vector-transfected cells (B).
Fig. 2.
Fig. 2.
CALHM1 controls ERK1/2 signaling. (A,B) HT-22 cells transiently transfected with CALHM1 or empty vector were challenged (A) or not (B) with Ca2+ add-back conditions for the indicated periods of time. Cell extracts were then analyzed by WB for the indicated proteins. C, CALHM1; V, vector. The arrowhead indicates phospho-Ser376-MSK1.
Fig. 3.
Fig. 3.
ERK1/2 signaling activation by CALHM1 is mediated by Ras. (A–D) HT-22 cells transiently transfected with CALHM1 or empty vector were pre-incubated for 30 min in the presence or absence (CTRL) of 10 µM KN62 or 5 µM KN93 (CaMKII inhibitors); 10 µM bisindolylmaleimide III (BisIII) or 1 µM Ro31-8220 (Ro31) (PKC inhibitors); 20 µM H89 or 0.8 µM PKI (PKA inhibitors); 20 µM PD98059 (PD98, MEK inhibitor) or 50 µM farnesyl thiosalicylic acid (FTS, Ras inhibitor). Cells were then challenged with Ca2+ add-back conditions for 10 min and cell extracts were analyzed by WB for the indicated proteins. C, CALHM1; V, vector. The arrowhead indicates phospho-MSK1. Experiments in A–D are representative of three independent experiments.
Fig. 4.
Fig. 4.
Effect of CALHM1-mediated Ca2+ influx inhibition on ERK1/2 signaling activation by CALHM1. (A) Free cytoplasmic Ca2+ measurements with Fluo-4 loading and Ca2+ add-back in HT-22 cells transiently transfected with CALHM1 or empty vector. Cells pre-incubated in Ca2+-free buffer (0 CaCl2), in either the presence or absence of 20 µM Ruthenium Red (RuRed), were challenged with physiological extracellular Ca2+ concentrations (1.4 mM CaCl2) to monitor the progressive restoration of cytoplasmic Ca2+ levels. Traces illustrate mean relative fluorescence units (RFU) of 2 independent measurements. (B,C) HT-22 cells transiently transfected with CALHM1 or empty vector were pre-incubated in the presence or absence (Basal and CTRL) of 20 µM Ruthenium Red (RuRed), 100 µM Gd3+, 20 µM Zn2+, or 20 µM BAPTA-AM. Cells were then challenged or not (Basal) with Ca2+ add-back conditions for 10 min and cell extracts were analyzed by WB for the indicated proteins. C, CALHM1; V, vector. The arrowhead indicates phospho-MSK1. Experiments in B and C are representative of three independent experiments. (D) Densitometric analyses and quantification of the phospho-ERK1/2/total ERK1/2 ratio (pERK/tERK) in CALHM1-transfected cells [expressed as a percentage of the ratio in corresponding vector-transfected control conditions (% of V)]. Error bars, s.e.m. [n = 3; *P<0.05 (versus Basal); #P<0.05 (versus CTRL); one-way ANOVA Bonferroni post-hoc tests].
Fig. 5.
Fig. 5.
N140A-, W114A-, and P86L-CALHM1 mutations affect CALHM1-mediated Ca2+ influx. (A,D,G) Cytoplasmic Ca2+ measurements with Fluo-4 loading and Ca2+ add-back in HT-22 cells transiently transfected with empty vector, Myc-tagged WT-CALHM1, and the indicated Myc-tagged CALHM1 mutants. Traces illustrate mean relative fluorescence units (RFU) of 3 independent measurements. (B,E,H) Peak of Ca2+ concentration measurements, as in A, D and G, respectively, expressed as ΔF/F0. Error bars, s.e.m. (B, n = 6; E, n = 7; H, n = 4; *P≤0.0002; Student's t-test with Bonferroni correction). (C,F,I) WB analysis of CALHM1 expression in the corresponding cell lysates. V, vector; WT, WT-CALHM1.
Fig. 6.
Fig. 6.
N140A-, W114A-, and P86L-CALHM1 mutations affect the control of ERK1/2 signaling activation by CALHM1. (A) HT-22 cells transiently transfected with empty vector (V), WT-CALHM1 (WT), and the indicated CALHM1 mutants were challenged with Ca2+ add-back conditions for 10 min. Cell extracts were then analyzed by WB for the indicated proteins. The arrowhead indicates phospho-MSK1. (B) Densitometric analysis and quantification of the ratio phospho-ERK2/total ERK2 (pERK2/tERK2). a.u., arbitrary units. Error bars, s.e.m. (n = 3; *P<0.005; **P<0.0001; Student's t-test with Bonferroni correction).
Fig. 7.
Fig. 7.
Primary neurons from Calhm1 KO mice display impaired ERK1/2 signaling activation. (A) Real time PCR analyzing Calhm1 expression levels in 14 DIV primary neurons from Calhm1 +/+ (WT) and −/− (KO) mice. Calhm1 expression was normalized to the reference genes HPRT1, TBP, and POLR2A. N.D., not detected. (B) 14 DIV primary neurons isolated from Calhm1 KO mice and WT littermate controls were challenged or not (Basal) with Ca2+ add-back conditions for the indicated periods of time. Cell extracts were then analyzed by WB for the indicated proteins. The arrowhead indicates phospho-MSK1. The figure shows representative results from four independent experiments.
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References

    1. Adams J. P., Sweatt J. D. (2002). Molecular psychology: roles for the ERK MAP kinase cascade in memory. Annu. Rev. Pharmacol. Toxicol. 42, 135–163. 10.1146/annurev.pharmtox.42.082701.145401 - DOI - PubMed
    1. Aguilar D., Skrabanek L., Gross S. S., Oliva B., Campagne F. (2008). Beyond tissueInfo: functional prediction using tissue expression profile similarity searches. Nucleic Acids Res. 36, 3728–3737. 10.1093/nar/gkn233 - DOI - PMC - PubMed
    1. Berridge M. J. (2012). Calcium signalling remodelling and disease. Biochem. Soc. Trans. 40, 297–309. 10.1042/BST20110766 - DOI - PubMed
    1. Boada M., Antúnez C., López–Arrieta J., Galán J. J., Morón F. J., Hernández I., Marín J., Martínez–Lage P., Alegret M., Carrasco J. M.et al. (2010). CALHM1 P86L polymorphism is associated with late-onset alzheimer’s disease in a recessive model. J. Alzheimers Dis. 20, 247–251. - PubMed
    1. Borst J. G., Sakmann B. (1999). Depletion of calcium in the synaptic cleft of a calyx-type synapse in the rat brainstem. J. Physiol. 521, 123–133. 10.1111/j.1469-7793.1999.00123.x - DOI - PMC - PubMed

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