Figure 3. CALHM1 controls Ca²⁺ influx by a mechanism that does not promote VGCC or SOCE channel activation.

(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).