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Results: 5

1.
Figure 1

Figure 1. Alignment and phylogeny of CALHM1. From: A polymorphism in CALHM1 influences Ca2+ homeostasis, A?levels, and Alzheimer's disease risk.

(A) Sequence alignment of human CALHM3, CALHM2, and CALHM1, and of murine and C. elegans CALHM1. Conserved sequences are highlighted in blue and sequence conservation is mapped in a color gradient, the darkest color representing sequences with absolute identity and lighter colors representing sequences with weaker conservation. Boxes denote hydrophobic domains 1–4 (HD1–4). Stars, predicted N-glycosylation sites on human CALHM1.
(B) Phylogenetic tree including human CALHM1 (hCALHM1).

Ute Dreses-Werringloer, et al. Cell. ;133(7):1149-1161.
2.
Figure 2

Figure 2. Tissue expression, subcellular localization, and N-glycosylation of human CALHM1. From: A polymorphism in CALHM1 influences Ca2+ homeostasis, A?levels, and Alzheimer's disease risk.

(A) Total RNA was used for RT-PCR analyses targeting CALHM1 and β-actin transcripts in multiple human tissues and brain regions.
(B) Immunofluorescence staining in CHO cells transfected with human Myc-tagged CALHM1 using anti-Myc (green) and anti-GRP78 (red) antibodies.
(C) Lysates from HT-22 cells transfected with wild type (WT) or mutated (N140A and N74A) Myc-CALHM1, were incubated in the absence (−) or presence (+) of endoglycosidase H (Endo H) or N-glycosidase F (PNGase F). Cell lysates were probed with anti-Myc (upper panels) and anti-actin (lower panels) antibodies.
(D) Cell surface-biotinylated proteins from Myc-CALHM1-transfected HT-22 cells were precipitated using immobilized avidin and probed with anti-Myc (upper panel) and anti-N-cadherin (lower panel, cell surface positive control) antibodies.

Ute Dreses-Werringloer, et al. Cell. ;133(7):1149-1161.
3.
Figure 3

Figure 3. CALHM1 controls Ca2+ influx by a mechanism that does not promote VGCC or SOCE channel activation. From: A polymorphism in CALHM1 influences Ca2+ homeostasis, A?levels, and Alzheimer's disease risk.

(A) Cytoplasmic Ca2+ measurements using Fluo-4 loading and Ca2+ add-back assays in HT-22 cells transiently transfected with Myc-CALHM1 or control vector. Cells were first incubated in Ca2+-free buffer (0 CaCl2) and then challenged with physiological extracellular Ca2+ concentrations (1.4 mM CaCl2) to monitor the progressive restoration of basal [Ca2+]i. Traces illustrate the mean relative fluorescence units (RFU) +/− S.D. (shaded areas) of three independent experiments. Inset, WB of the corresponding cell lysates probed with anti-Myc antibody (Vec, vector; C, CALHM1).
(B) Peak and steady-state of [Ca2+]i measurements as in (A) expressed in ΔF/F0 (*, P<0.001; Student’s t test).
(C–H) Cytoplasmic Ca2+ measurements as in (A) in cells pretreated with 2-APB [50 μM, (C)], SNX-482 [0.5 μM, (D)], mibefradil [1 μM, (D)], nifedipine [10 μM, (E)], ω-conotoxin MVIIC [Conotoxin, 5 μM, (E)], dantrolene [DTL, 10 μM, (F)], xestospongin C [XeC, 2 μM, (F)], or with the indicated concentrations of CoCl2 (G) and NiCl2 (H). Traces in (C–H) illustrate representative measurements of 2–3 independent experiments.
(I) WB with anti-Myc (upper panels) and anti-actin (lower panels) antibodies of protein extracts obtained from cells treated as in (G) and (H).

Ute Dreses-Werringloer, et al. Cell. ;133(7):1149-1161.
4.
Figure 4

Figure 4. Ion channel properties of CALHM1. From: A polymorphism in CALHM1 influences Ca2+ homeostasis, A?levels, and Alzheimer's disease risk.

(A) Lysates from non-transfected (NT) and Myc-CALHM1-tranfected HEK293 cells were analyzed by WB in the absence (Control) or presence of β-mercaptoethanol (+βME) using anti-Myc (two upper panels) and anti-actin (lower panel) antibodies.
(B) Lysates from HEK293 cells transfected (+) or not (−) with V5-tagged CALHM1 (V5-CALHM1) or Myc-CALHM1, were immunoprecipitated with anti-Myc antibody. Total lysates (Input, left panels) and immunoprecipitates (Anti-Myc IP, right panels) were analyzed by WB using antibodies against V5 (upper panels), Myc (middle panels), and actin (lower panels).
(C) Partial sequence alignment of human NMDAR NR2 (NMDAR2) subunits A–D and CALHM1 from various species. Sequence conservation is highlighted in a blue gradient as described in Fig. 1A. Star denotes Q/R/N site.
(D) Cytoplasmic Ca2+ measurements in HT-22 cells transiently transfected with control vector and WT or N72G-mutated Myc-CALHM1. Cells were treated and results analyzed as in Fig. 3A (n = 3 independent experiments). Inset, WB of the corresponding cell lysates with anti-Myc antibody.
(E) Peak of [Ca2+]i measurements as in (D) expressed in ΔF/F0 (*, P<0.001; Student’s t test).
(F) Representative current traces during voltage ramps in Xenopus oocytes injected with CALHM1 cRNA (blue and green traces) or water (red trace) in normal LCa96 solution (blue and red traces) or in Na+-free LCa96 solution (replaced with equimolar N-methyl-D-glucamine (NMDG); green trace).
(G) Whole-cell currents in CALHM1-expressing (blue and red traces) or control (black trace) CHO cells in response to voltage ramps before (blue trace) and after (red trace) perfusion with 100 μM Gd3+. Bath contained 120 mM NaCl, pipette solution contained 122 mM CsCl (see Experimental Procedures). Cell capacitances of the CALHM1-expressing and control cells were 18.5 pF and 13.0 pF, respectively.
(H) Whole-cell currents in CALHM1-expressing CHO cells (uncorrected for leakage currents) in response to voltage ramps in bi-ionic Ca2+/Cs+ solutions (20 mM Ca-aspartate in bath, 120 mM Cs-aspartate in pipette; see Experimental Procedures) before (blue trace) or after (red trace) bath addition of 100 μM Gd3+(Cm = 24.1 pF). Reversal potential Vrev = +8.3 ± 2.9 mV (n = 7) after correction for liquid junction potential and leakage current, indicating PCa : PCs = 5. No currents were observed in CALHM1-expressing cells with NMDG-aspartate in bath and pipette solutions (black trace; Cm = 20.5 pF).

Ute Dreses-Werringloer, et al. Cell. ;133(7):1149-1161.
5.
Figure 5

Figure 5. The CALHM1 P86L polymorphism influences Ca2+ homeostasis, APP processing, and AD risk. From: A polymorphism in CALHM1 influences Ca2+ homeostasis, A?levels, and Alzheimer's disease risk.

(A and B) SwAPP695-N2a cells were transiently transfected with control vector or with WT or P86L-mutated Myc-CALHM1. Six and half hours post-transfection, medium was changed and cells were incubated for 60 min in the absence or presence of Ca2+ add-back conditions as described in Experimental Procedures. Total secreted Aβ and sAPPα, and cellular APP and Myc-CALHM1 were analyzed by WB (A). Secreted Aβ1-40 and Aβ1-42 were analyzed by ELISA in the presence of Ca2+ add-back conditions (n = 12; Student’s t test) (B).
(C–E) APP695-SH-SY5Y cells differentiated for 15 days with retinoic acid were treated for 3 days with Accell siRNAs directed against human CALHM1. Medium was then changed and cells were incubated for 90 min in the absence or presence of Ca2+ add-back conditions. Total secreted Aβ and cellular APP and actin were analyzed by WB (C). Total secreted Aβ1-x was quantified by ELISA (n = 3; Student’s t test) (D). CALHM1 mRNA levels were assayed by real-time qRT-PCR analysis. Histogram illustrates the mean relative CALHM1 expression ± S.D. (control, n = 4; CALHM1 siRNA, n = 3) (E).
(F) Five independent case-control studies were analyzed to assess the association of rs2986017 with AD risk. The allelic OR (T vs. C) was estimated in each population and in the combined one. 1Test for heterogeneity: χ2 = 2.84, df = 4, P = 0.59; Test for overall effect: Z = 6.06, P = 2.10−9 (Mantel-Haentzel method, fixed OR = 1.42 [1.27–1.59]).
(G) Whole-cell currents in CHO cells expressing WT- (blue trace; Cm = 13.2 pF) or P86L-CALHM1 (green trace; Cm = 22.9 pF) in same bi-ionic conditions as in Fig. 4H. P86L-CALHM1-expressing cells remained sensitive to block by 100 μM Gd3+ (red trace), but the reversal potential was shifted to more hyperpolarized voltages (Vrev = −8.9 ± 3.6 mV ; n = 6), indicating a reduced Ca2+ permeability (PCa : PCs = 2) compared with that of WT-CALHM1.
(H) Cytoplasmic Ca2+ measurements in HT-22 cells transiently transfected with control vector and WT or P86L-mutated Myc-CALHM1. Cells were treated and results analyzed as in Fig. 3A (n = 3 independent experiments). Inset, WB of the corresponding cell lysates with anti-Myc antibody. (I) Peak of [Ca2+]i measurements as in (H) expressed in Δ F/F0 (*, P<0.001; Student’s t test).

Ute Dreses-Werringloer, et al. Cell. ;133(7):1149-1161.

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