α-Latrotoxin Actions in the Absence of Extracellular Ca2+ Require Release of Stored Ca2

Abstract

α-Latrotoxin (αLTX) causes exhaustive release of neurotransmitters from nerve terminals in the absence of extracellular Ca²⁺ (Ca²⁺_e). To investigate the mechanisms underlying this effect, we loaded mouse neuromuscular junctions with BAPTA-AM. This membrane-permeable Ca²⁺-chelator demonstrates that Ca²⁺_e-independent effects of αLTX require an increase in cytosolic Ca²⁺ (Ca²⁺_cyt). We also show that thapsigargin, which depletes Ca²⁺ stores, induces neurotransmitter release, but inhibits the effect of αLTX. We then studied αLTX's effects on Ca²⁺_cyt using neuroblastoma cells expressing signaling-capable or signaling-incapable variants of latrophilin-1, a G protein-coupled receptor of αLTX. Our results demonstrate that αLTX acts as a cation ionophore and a latrophilin agonist. In model cells at 0 Ca²⁺_e, αLTX forms membrane pores and allows the influx of Na⁺; this reverses the Na⁺-Ca²⁺ exchanger, leading to the release of stored Ca²⁺ and inhibition of its extrusion. Concurrently, αLTX stimulates latrophilin signaling, which depletes a Ca²⁺ store and induces transient opening of Ca²⁺ channels in the plasmalemma that are sensitive to inhibitors of store-operated Ca²⁺ entry. These results indicate that Ca²⁺ release from intracellular stores and that Ca²⁺ influx through latrophilin-activated store-operated Ca²⁺ channels contributes to αLTX actions and may be involved in physiological control of neurotransmitter release at nerve terminals.

Keywords: ADGRL1; calcium; intracellular Ca2+ stores; latrophilin-1; neuroblastoma cells; neuromuscular junction; neurotransmitter release; store-operated Ca2+ entry; α-Latrotoxin.

Figure 1

αLTX actions do not require extracellular Ca²⁺ but strictly depend on intracellular Ca²⁺. (a) Examples of the effect of 0.5 nM αLTX on the frequency of spontaneous MEPPs in mouse neuromuscular preparations in the absence of Ca²⁺_e, continuously recorded from individual muscle fibers. (b) The control experimental protocol: initial incubation with 2 mM Ca²⁺_e; removal of Ca²⁺_e; addition of 0.5 nM αLTX; reintroduction of 2 mM Ca²⁺_e. (c) Representative Vm recordings during respective experimental stages. (d,e) Mean MEPP frequencies and amplitudes during the experimental stages as indicated below. (f) The Ca²⁺_cyt chelation protocol: initial incubation in a Ca²⁺_e-free buffer; incubation with 200–500 μM BAPTA-AM; two extended washing steps with a Ca²⁺-free buffer; addition of 0.5 nM αLTX; reintroduction of 2 mM Ca²⁺_e. (g) Representative Vm recordings under the experimental conditions indicated. (h,i) Mean MEPP frequencies and amplitudes during respective experimental stages. The bars are the means ± SEM; the bar colors correspond to protocol phases; the underlying data points are shown as white circles; asterisks show statistical significance compared to Ca²⁺_e-free control, unless indicated by lines; the blue asterisk in (h) compares the values indicated by the two blue bars; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, non-significant; for each condition shown, n = 9–43 individual muscle fibers, from 3 to 4 independent neuromuscular preparations.

Figure 2

The depletion of intracellular Ca²⁺ stores inhibits the Ca²⁺_e-independent actions of αLTX. (a) An example of the effect of TG and subsequent αLTX on the frequency of spontaneous MEPPs. The experimental protocol shown above the trace included the following phases: initial incubation in a Ca²⁺_e-free buffer; addition of 10 μM TG; addition of 0.5 nM αLTX; and reintroduction of 2 mM Ca²⁺_e. (b) Representative Vm recordings during respective experimental stages. (c) Average MEPPs in the absence and presence of TG, in the absence of Ca²⁺_e. (d,e) Mean MEPP amplitudes and half-widths during the indicated experimental stages. (f) Mean MEPP frequencies’ respective experimental stages. The bars are the means ± SEM; the bar colors correspond to protocol phases; asterisks show statistical significance compared to Ca²⁺_e-free control; the underlying data points are shown as white circles; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, non-significant; n = 12–36 individual muscle fibers from 4 to 7 independent neuromuscular preparations.

Figure 3

ADGRL1 constructs expressed in NB2a cells specifically bind αLTX. (a) The structures and membrane topologies of the two ADGRL1 constructs. (b) Stable expression of the ADGRL1 constructs in NB2a cells. Whole-cell lysates were separated by 8% SDS-PAGE, blotted and probed with an anti-V5 antibody. Top, a typical Western blot representative of four independent experiments. Bottom, quantification of the expression data; n = 4. NB2a, un-transfected cells. (c) The expressed ADGRL1 constructs bind αLTX. Transfected cells were incubated with 5 nM αLTX and centrifuged. The supernatants and cell pellets were separated by SDS-PAGE, blotted and probed with an anti-LTX antibody. Top, a representative Western blot showing unbound αLTX in the supernatants and receptor-bound αLTX in the cell pellets. Bottom, relative amounts of αLTX bound to respective cells; n = 3. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Figure 4

αLTX releases intracellular Ca²⁺ and triggers vast Ca²⁺ influx in cells expressing full-size receptors only. (a) A representative trace of changes in Ca²⁺ fluorescence in Fluo-4-loaded NB2a cells treated with 0.3 μM TG. The following characteristic stages are indicated: in the absence of Ca²⁺_e, TG induces intracellular Ca²⁺ release; reintroduction of Ca²⁺_e leads to a transient Ca²⁺ influx (Ca²⁺ peak) and Ca²⁺_cyt equilibrium (Ca²⁺ Eq). (b) Representative fluorescence traces showing αLTX-mediated effects in control cells and cells expressing LPH. (c) Relative Ca²⁺ release measured at end of Ca²⁺_e-free period. (d) Amplitude of transient Ca²⁺ peak on the addition of Ca²⁺_e. (e) Relative Ca²⁺_cyt Eq in the presence of Ca²⁺_e. Bars show the means ± SEM from 3 to 7 independent experiments performed in triplicate. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; NS, non-significant.

Figure 5

αLTX-mediated Ca²⁺_cyt regulation involves signaling and non-signaling mechanisms. (a) LPH- and ΔLPH-expressing NB2a cells were incubated in Ca²⁺-free buffer, then treated with 1 nM αLTX, and exposed to 2 mM Ca²⁺_e. The fluorescence traces shown are the averages of three replicates and representative of four independent experiments. (b) Initial rate of intracellular Ca²⁺ release. (c) Relative Ca²⁺ release at the end of Ca²⁺_e-free period. (d) Amplitude of the transient Ca²⁺ influx peak in the presence of Ca²⁺_e. (e) Relative Ca²⁺ Eq in the presence of Ca²⁺_e. Bars show the means ± SEM; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; NS, non-significant.

Figure 6

In model cells, αLTX does not release Ca²⁺ from the ER. (a) LPH- and ΔLPH-expressing NB2a cells were treated with 0.3 μM TG, then stimulated with 1 nM αLTX and exposed to 2 mM Ca²⁺_e. Representative fluorescence traces show the averages of three replicates. (b–e) Ca²⁺_cyt changes relative to the αLTX-induced effects in the LPH-expressing cells (blue bars). (b) Amplitudes of αLTX-mediated Ca²⁺ release. (c) Rates of Ca²⁺ release during the Ca²⁺-free period. (d) Amplitudes of transient Ca²⁺ influx in the presence of Ca²⁺_e. (e) Levels of Ca²⁺_e Eq. Asterisks show statistical significance compared to LPH-cells + αLTX (blue bars), other comparisons are shown by horizontal lines. Bars are the means ± SEM (n = 3–4); *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; NS, non-significant.

Figure 7

The αLTX pore regulates [Ca²⁺]_cyt by inducing Na⁺ influx, while receptor-mediated action does not require Na⁺ influx. (a) LPH- and ΔLPH-expressing NB2a cells were incubated in buffer containing Na⁺ or the Na⁺ substitute NMDG, then stimulated with 1 nM αLTX and exposed to 2 mM Ca²⁺_e. Representative fluorescence traces show the averages of three replicates. (b–e) Ca²⁺_cyt changes relative to the αLTX-induced effects in the LPH-expressing cells in the presence of Na⁺. (b) Amplitudes of αLTX-mediated Ca²⁺ release. (c) Amplitudes of transient Ca²⁺ influx peaks. (d) Levels of Ca²⁺_e Eq at the end of experiment. The bars are the means ± SEM (n = 2–5); the asterisks show statistical significance compared to the LPH/αLTX/Na⁺ condition, other comparisons are shown by horizontal lines. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-significant.

Figure 8

SKF inhibits αLTX-induced Ca²⁺ release and subsequent Ca²⁺ influx. (a) LPH-expressing NB2a cells were incubated in buffer containing 145 mM Na⁺ or 145 mM NMDG. The cells were then treated with 100 μM SKF, stimulated with 1 nM αLTX and exposed to 2 mM Ca²⁺_e. The fluorescence traces are the averages of three replicates and are representative of five independent experiments. (b–e) Ca²⁺_cyt changes relative to the αLTX-induced effects in the LPH-expressing cells in the presence of Na⁺. (b) SKF-induced Ca²⁺ release. (c) αLTX-mediated Ca²⁺ release with and without prior SKF treatment. (d) Amplitude of the Ca²⁺ peaks under respective conditions. (e) Ca²⁺ Eq levels after respective treatments. The bars are the means ± SEM (n = 5); the asterisks show statistical significance compared to LPH/Na⁺/αLTX, other comparisons are shown by horizontal lines; *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, non-significant.

Figure 9

Dual actions of αLTX on Ca²⁺_cyt in presynaptic nerve terminals that lead to neurotransmitter release. (a) Idealized Ca²⁺_cyt dynamics induced by TG and the two LTX actions (after subtracting control traces). (i) TG blocks SERCA and causes Ca²⁺ release from the ER, which activates a transient SOCE upon reintroduction of Ca²⁺_e; Ca²⁺_cyt decays due to NCX activity. (ii) Combined αLTX activity, consisting of the ADGRL1- and pore-mediated effects. (iii) Receptor-dependent αLTX action calculated by subtracting the αLTX pore-mediated effect (iv) from the combined αLTX actions (ii). Receptor-mediated signaling causes a slow release of Ca²⁺ from intracellular stores and subsequent opening of a large pool of SOCCs. Reintroduction of Ca²⁺_e leads to a transient SOCE that exceeds that caused by TG. ADGRL1 signaling also opens non-inactivating Ca²⁺ channels that contribute to the elevated Ca²⁺_cyt after SOCE. (iv) αLTX pore-mediated effects based on our experiments with ΔLPH and NMDG. αLTX pores mediate the influx of Na⁺_e, which reverses NCX. Reversal of NCX located on the Ca²⁺ stores (ER and MC) and cell membrane leads to a slow increase in [Ca²⁺]_cyt, and inhibits Ca²⁺_cyt extrusion. Upon reintroduction of Ca²⁺_e, Ca²⁺_e influx via αLTX pores and inhibition of Ca²⁺_cyt extrusion elevate the [Ca²⁺]_cyt further. (b) A model of αLTX action. αLTX binds and activates ADGRL1, and forms pores in the cell membrane. Na⁺ and Ca²⁺ enter though the αLTX pores. Elevated [Na⁺]_cyt reverses NCX located on the MC, ER, and cell membrane. This releases Ca²⁺ from the MC and ER, and inhibits Ca²⁺_cyt extrusion. The pore-mediated [Ca²⁺]_cyt increase triggers exocytosis of synaptic vesicles (SV). αLTX also activates G protein signaling via ADGRL1, resulting in Ca²⁺ release from the ER and/or other stores, and the activation of SOCCs (via store depletion and/or by direct signaling). However, ADGRL1-mediated αLTX action requires Ca²⁺ influx via SOCCs to develop its full effect and cause a burst-like release of SVs. IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptor; NCLX, Na⁺-Ca²⁺ exchanger of the internal MC membrane; STIM, proteins detecting Ca²⁺ release from ER; SV, synaptic vesicles.