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PLoS One
2014 Nov 06;911:e112484. doi: 10.1371/journal.pone.0112484.
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Effect of the CALHM1 G330D and R154H human variants on the control of cytosolic Ca2+ and Aβ levels.
Vingtdeux V
,
Tanis JE
,
Chandakkar P
,
Zhao H
,
Dreses-Werringloer U
,
Campagne F
,
Foskett JK
,
Marambaud P
.
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CALHM1 is a plasma membrane voltage-gated Ca2+-permeable ion channel that controls amyloid-β (Aβ) metabolism and is potentially involved in the onset of Alzheimer's disease (AD). Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant (minor allele frequency (MAF) = 0.015%), but that G330D is not (MAF = 3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca2+ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca2+ uptake and Aβ levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca2+ and Aβ accumulation. While these results strengthen the notion that CALHM1 influences Aβ metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.
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Figure 2. Effect of the CALHM1 G330D and R154H variants on Ca2+ influx in mammalian cells.A. Ca2+i measurements with Fluo-4 loading and Ca2+ add-back in HT-22 cells transiently transfected with WT-, G330D-, R154H-, and W114A-CALHM1 or empty vector. Cells were incubated in Ca2+-free buffer (0 CaCl2) for 30 min, and then challenged with physiological Ca2+o concentration (1.4 mM CaCl2) to monitor the restoration of Ca2+i levels. RFU, relative fluorescence units. B. Peak of Ca2+i concentration measurements after Ca2+ add-back expressed as ÎF/F0. Error bars, SEM. *P<0.01, **P<0.001, relative to vector-transfected cells; #P<0.01, ##P<0.001, relative to WT-CALHM1-transfected cells (ANOVA with Bonferroni correction, nâ=â3 independent experiments as in (A)).
Figure 3. Effect of CALHM1 activation on Aβ levels.AâD. APP-N2a (A and B) and APP-HEK293 (C and D) cells transfected with empty vector or WT-CALHM1 were subjected to Ca2+ add-back conditions in the absence (CaAB, A and C) or presence of 1% FBS (CaAB 1% FBS, B and D). Extracellular Aβ and sAPPα were analyzed by WB after the indicated periods of secretion. Cell lysates were probed using anti-Myc and anti-actin antibodies to detect CALHM1 and actin, respectively.
Figure 4. Effect of the CALHM1 G330D and R154H variants on Aβ levels.A. APP-N2a cells were transiently transfected with empty vector or WT-, G330D-, and R154H-CALHM1, as well as W114A-CALHM1 (CALHM1 dead mutant control, see Ref. [13]). Cells were then subjected to Ca2+ add-back as in Fig. 3A. Extracellular Aβ and sAPPα were analyzed by WB after 1 hr of secretion. Cell lysates were probed using anti-Myc, anti-BACE1, and anti-actin antibodies to detect CALHM1, BACE1, and actin, respectively. B. Densitometric analyses and quantification of Aβ levels in 3 independent measurements as in (A). Error bars, SEM; *P<0.01, **P<0.001, relative to vector-transfected cells; #P<0.05, ##P<0.001, relative to WT-CALHM1-transfected cells (ANOVA with Bonferroni correction).
Figure 1. Effect of the R154H and G330D variants on CALHM1 gating and Ca2+ permeability.
A–C. Currents observed in oocytes expressing (A) WT-CALHM1, (B) G330D-CALHM1, and (C) R154H-CALHM1 in standard bath solution containing 2 mM Ca2+
o in response to voltage pulses from −80 mV to +60 mV; holding potential −40 mV. D. Following a series of voltage pulses, currents at −80 mV were measured to determine G-V relations in the presence and absence of Ca2+
o. For each oocyte, Gmax was determined by fitting 0 mM Ca2+
o data with a Boltzmann function; all currents were then normalized to Gmax. Normalized data were fit with Boltzmann functions with the assumption that Ca2+ does not affect Gmax
[14], [15]. WT-CALHM1 0 mM Ca2+
o (red circles) V0.5 =  −75.9 mV; R154H-CALHM1 0 mM Ca2+
o (blue circles) V0.5 =  −82.8 mV; G330D-CALHM1 0 mM Ca2+
o (black circles) V0.5 =  −78.8 mV; WT-CALHM1 2 mM Ca2+
o (red triangles) V0.5 = 60.8 mV; R154H-CALHM1 2 mM Ca2+
o (blue triangles) V0.5 = 62.4 mV; G330D-CALHM1 2 mM Ca2+
o (black triangles) V0.5 = 62.8 mV. E. Changes in Erev resulting from changing from 0 to 2 mM Ca2+
o solution in oocytes expressing WT-CALHM1 (red), G330D-CALHM1 (black), and R154H-CALHM1 (blue). n = 4–6 oocytes for each condition; Error bars, SE.
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