The Mechanism of LTXN4C-Induced Ca2+ Influx Involves Latrophilin-Mediated Activation of Cav2.x Channels

Figures

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  Figure 1. Changes in cell morphology and LPHN1 expression with cell differentiation. (a) Wild-type and mutant receptor constructs that were used to stably transfect NB cells. LPHN1 is self-cleaved into two fragments (NTF and CTF). The mutant construct ΔLPH, consisting of the NTF and a C-terminal segment of neurexin I (NRX), is also self-cleaved. The NTF and CTF contain immunological tags (V5 and myc) for reliable detection. (b) A shift to neuron-like morphology induced in NB, LPH, and ΔLPH cells by construct expression and differentiation. The cells were maintained in complete medium or differentiated by 48 h incubation in SF medium ± 1 mM dbcAMP (dbcA). Neuron-like (N-type) cells have compact somata and show high phase contrast (white halo). The scale bars are 30 μm. (c) Per cent of N-type cells in each culture upon differentiation under respective conditions. (d) Expression of the receptor constructs in stably transfected LPH and ΔLPH cells. NB, LPH, and ΔLPH cells were differentiated by serum deprivation for 24 h, fixed, and labeled with an anti-V5 antibody and Alexa Fluor-488-conjugated IgG (green). The scale bar is 30 μm. (e) LPH cells overexpress LPHN1 upon differentiation. LPH cells…

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  Figure 2. LTXN4C induces an increase in [Ca2+]cyt via LPHN1 but does not involve the TG-sensitive Ca2+ stores. LPH-SF cells were loaded with the Ca2+-sensing dye Fluo-4 AM and recorded in a multi-well fluorescent plate reader, and stimulated as shown above the traces (a,c). The dye-loading protocol, applicable to all experiments with Fluo-4, is described in detail in Section 4.7.1. Initial incubation in a nominally Ca2+-free medium was followed by the addition of LTXN4C or TG, and subsequent addition of 2 mM Ca2+. (a) Dose dependence of changes in Ca2+cyt fluorescence induced by 0–3 nM LTXN4C. (b) Quantification of the data in (a) after subtracting the basal Ca2+ fluorescence. LTXN4C causes a small increase in Ca2+cyt in the Ca2+-free buffer and a large Ca2+e influx after Ca2+ addition. Both effects are LTXN4C dose-dependent. (c) LTXN4C and TG induce Ca2+ release and influx by acting through different mechanisms. Normalized Ca2+ fluorescence changes triggered by 1 nM LTXN4C before or after treatment with 0.3 μM TG in the absence and then presence of Ca2+e. Buf, buffer (no stimulus). The main phases of TG action are also indicated by arrows: Ca2+ release, SOCE peak, and post-SOCE Ca2+cyt…

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  Figure 3. Changes in Ca2+cyt levels in individual NB, LPH, and ΔLPH cells upon stimulation with LTXN4C. The cells were loaded with the Ca2+ sensing dye Fluo-4 AM, recorded under a confocal microscope, and stimulated as indicated by bars at the top. (a,b) The cells were incubated in a Ca2+-free medium, when the background fluorescence F0 was recorded. The cells were then stimulated with 2 nM LTXN4C, supplied with 2 mM Ca2+e, and finally permeabilized with 2 nM αLTX to record the maximal Ca2+ fluorescence Fmax (arrowheads). All data were normalized to F0 and Fmax, as described in Section 4.7.2. (a) Average traces ± SE from 22 to 26 individual NB, LPH and ΔLPH cells. RFU, normalized relative fluorescence units. (b) Individual fluorescence traces ± SE from randomly selected LPH cells. The data are representative of n = 4 independent experiments with 20–30 individual cells recorded in each (N = 80–120). (c–h) The LPH cells were incubated in the presence of 2 mM Ca2+e, stimulated with 2.5 nM LTXN4C, and permeabilized with 2.5 nM αLTX (arrowhead). (c) Selected time-lapse fluorescent images of a group of cells (1–4). The numbers indicate the time in seconds from the beginning of recording; the…

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  Figure 4. Expression of SOCE-associated proteins in proliferating and differentiated NB, LPH, and ΔLPH cells. (a) Agarose gel analysis of RT-PCR products obtained on cDNA prepared from mouse forebrain, the ventral horn of the lumbar segment of mouse spinal cord (SC VH), NB cells (NB), and two receptor-expressing cell lines (LPH and ΔLPH), which were proliferating (PC) in complete medium or differentiated for 48 h in SF medium (SF). The following SOCE-associated mRNAs were detected (expected band size, bp): STIM1 (296), STIM2 (256), SARAF (280), Orai1 (335), Orai2 (327), Orai3 (318), and TRPC2 (181). TRPC6 (179) was detected in LPH cells only. The arrowheads show the low levels of TRPC1, 3 and 6 in the spinal cord. An additional band found in all reactions targeting STIM1 cDNA did not affect the relative quantification of STIM1. PC: proliferating cells; SF: cells differentiated in SF medium. Numbers on the left show the sizes of selected markers in bp. The gels are representative of n = 3 experiments with 3 replicates (N = 9), which gave similar results. (b–d) Relative quantification of SOCE proteins mRNA levels based on the results of qRT-PCR. (b) Fold-changes in mRNA levels induced by LPH…

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  Figure 5. Knockdown of Orai2 mRNA decreases Ca2+cyt levels during the Ca2+ release and influx phases. (a) The effect of sh1 or sh2 shRNA expression on the mRNA levels of SOCE-associated proteins. LPH cells were transfected with the sh1 or sh2 plasmid, allowed to grow for 24 h, and then differentiated in SF medium for 48 h. The mRNA levels were determined using qRT-PCR and normalized to β-actin and untransfected (control) values for each mRNA. Non-significant differences are not shown for simplicity. (b–d) The effect of Orai2 knockdown on stimulated changes in Ca2+cyt. To restrict Ca2+ fluorescence detection to knockdown cells, LPH cells were co-transfected with the sh2 and GCaMP6S plasmids. Subsequently, cells were grown for 24 h, differentiated in SF medium for 48 h, and stimulated (as shown by the bars above) with 0.3 μM TG (b), 1 nM αLTX (c), or 2 nM LTXN4C (d). Finally, the cells were incubated in 2 mM Ca2+e. (Left panels): Averaged traces of specific changes in Ca2+cyt fluorescence after the subtraction of basal fluorescence and normalization to baseline (Figure S5e). The dashed lines indicate 0. (Right panels): Fold-changes in aggregated specific Ca2+ fluorescence (area under the…

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  Figure 6. Knockdown of STIM2 mRNA downregulates Ca2+ stores but increases SOCE. (a–e) LPH cells were transduced with the lentivirus encoding anti-STIM2 shRNA at MOI = 60, allowed to grow for 24 h, differentiated in SF medium for 48 h, then loaded with Fluo-4 AM, and stimulated according to the standard protocol (shown above the traces), while recording the fluorescence response. (a) The effect of lentiviral shRNA transduction on mRNA levels of the main SOCE-associated proteins. The mRNA levels were determined using qRT-PCR and normalized to β-actin and control values for each protein (knockdown/control). (b–e) The effect of STIM2 knockdown on changes in Ca2+cyt in response to stimulation with 1 nM αLTX or 3 nM LTXN4C. (b,d) Time-courses of specific (above background) changes in Ca2+cyt fluorescence after normalization to baseline and subtraction of basal fluorescence. The dashed lines indicate 0. (c,e) Fold-change in aggregated (AUC) specific Ca2+ fluorescence in STIM2-knockdown cells relative to control cells during the Release, Influx/SOCE, and PostEq phases. For illustration, ΔFn values were aggregated over time for each phase as AUCs. The dashed lines indicate the control level…

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  Figure 7. VGCC α1-subunits expression in proliferating and differentiated NB and LPH cells. NB and LPH cells were maintained in complete medium or differentiated in SF medium for 48 h. mRNA was isolated from mouse brain and the proliferating or differentiated cell cultures, reverse transcribed, and used to amplify the fragments of respective VGCC α1-subunit by qRT-PCR employing specific primers. (a) The amplification products were analyzed by agarose gel electrophoresis. Fragments of the following sizes (in bp) were expected: Cav1.1 (334), Cav1.2 (353), Cav1.3 (337), Cav1.4 (260), Cav2.1 (253), Cav2.2 (354), Cav2.3 (376), Cav3.1 (246), Cav3.2 (230), Cav3.3 (471). PC: proliferating cells; SF: cells differentiated in SF medium. Numbers on the left show the sizes of selected markers in bp. The gels are representative of n = 3 experiments with 3 replicates (N = 9), which gave similar results. (b) Fold changes in VGCC mRNA levels induced by receptor expression in proliferating LPH cells, relative to proliferating NB cells. (c) Fold changes in VGCC mRNA levels induced by SF differentiation of NB cells, relative to proliferating NB cells. (d) Fold changes in VGCC mRNA levels induced by SF…

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  Figure 8. ω-Conotoxin MVIIC blocks LTXN4C-induced LPHN1-mediated SOCE/CICR. LPH-SF cells were incubated in Ca2+-free medium, exposed to 5 μM MVIIC or buffer (Control), then stimulated by 1 nM LTXN4C or treated with buffer, and finally supplied with 2 mM Ca2+e, as shown by bar above. (a) Changes in Ca2+ fluorescence under basal conditions or induced by LTXN4C in the absence (left) or presence (right) of MVIIC. The dashed lines represent zero. (b) Fold-changes in Ca2+ release (left) and Ca2+ influx (right) relative to buffer in the absence (Control) or presence of MIIC. The data are the means ± SE of n = 4 experiments (N = 12). Asterisks indicate the significance of differences (tested by FANOVA) between the connected values: *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, nonsignificant.