A Ca2+-independent receptor for alpha-latrotoxin, CIRL, mediates effects on secretion via multiple mechanisms

Abstract

alpha-Latrotoxin (alpha-Ltx), a component of black widow spider venom, stimulates secretion from nerve terminals and from PC12 cells. In this study we examine the effects of expression of a newly cloned Ca2+-independent receptor for alpha-Ltx (CIRL) on secretion from bovine chromaffin cells. We first characterized the effect of alpha-Ltx on secretion from untransfected cells. alpha-Ltx, by binding in a Ca2+-independent manner to an endogenous receptor, causes subsequent Ca2+-dependent secretion from intact cells. The stimulation of secretion is correlated with Ca2+ influx caused by the toxin. In permeabilized cells in which the Ca2+ concentration is regulated by buffer, alpha-Ltx also enhances Ca2+-dependent secretion, indicating a direct role of the endogenous receptor in the secretory pathway. Expression of CIRL increased the sensitivity of intact and permeabilized cells to the effects of alpha-Ltx, demonstrating that this protein is functional in coupling to secretion. Importantly, in the absence of alpha-Ltx, the expression of CIRL specifically inhibited the ATP-dependent component of secretion in permeabilized cells without affecting the ATP-independent secretion. This suggests that this receptor modulates the normal function of the regulated secretory pathway and that alpha-Ltx may act by reversing the inhibitory effects of the receptor.

Fig. 1.

α-Latrotoxin stimulates catecholamine secretion and Ca²⁺ uptake in cultured chromaffin cells. Monolayer cultures of bovine adrenal chromaffin cells were labeled with³H-norepinephrine (³H-NE), rinsed, and incubated with the indicated concentrations of α-Ltx in physiological saline without Ca²⁺ or Mg²⁺ and with 0.1 mm EGTA for 4 min. The toxin was removed, and the cells were incubated for 6 min in PSS containing 2.2 mmCa²⁺ and 0.5 mm Mg²⁺. The incubation solution was removed, the cells were lysed with 1% Triton X-100, and the amount of ³H-NE was determined by liquid scintillation spectrometry; n = 3 wells/group. Parallel cultures that had not been labeled with³H-NE also were incubated with α-Ltx, followed by an incubation in PSS containing 2.2 mmCa²⁺, 0.5 mm Mg²⁺, and 3 μCi/ml ⁴⁵Ca²⁺. After 4 min, the cells were rinsed immediately three times in PSS, and the amount of⁴⁵Ca²⁺ in the cells was determined by liquid scintillation spectrometry; n = 4 wells/group.

Fig. 2.

Expression of CIRL increases the sensitivity of intact chromaffin cells to stimulation by α-Ltx. Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7, which encodes a newly cloned protein that binds α-Ltx (CIRL), or pCMVneo (a control) by calcium phosphate precipitates, as described. After 4 d the cells were incubated with the indicated concentrations of α-Ltx in PSS without Ca²⁺ or Mg²⁺ and with 0.1 mm EGTA. After 4 min, the toxin was removed, and the cells were incubated for an additional 5 min in PSS containing 2.2 mm Ca and 0.5 mm Mg. The amounts of hGH (A) and catecholamine (B) released into the medium and the amounts remaining in the cells were determined as described; n = 4 wells/group.

Fig. 3.

Secretion stimulated by α-latrotoxin requires extracellular Ca²⁺. Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7 or pCMVneo, as in Figure 2. After 4 d the cells were incubated for 10 min with or without 26 pm α-Ltx in either PSS without Ca²⁺ or Mg²⁺ and with 0.2 mm EGTA (indicated as −Ca), or PSS containing 2.2 mm Ca²⁺ and 0.5 mmMg²⁺ (indicated as +Ca). The amounts of hGH (A) and catecholamine (B) released into the medium and the amounts remaining in the cells were determined as described; n = 4 wells/group.

Fig. 4.

CIRL supports both Ca²⁺ entry and the sustained elevation of intracellular Ca²⁺elicited by α-latrotoxin. A, HEK293 cells were transfected with plasmids for green fluorescent protein (GFP) and either pCDR7 (CIRL) or pCMVneo by Lipofectamine, as described. After 2 d the cells were incubated with the indicated concentrations of α-Ltx in physiological saline without Ca²⁺ or Mg²⁺ and with 0.2 mm EGTA for 4 min. The incubation with toxin was followed by a 6 min incubation in PSS containing 2.2 mm Ca²⁺, 0.5 mm Mg²⁺, and 1 μCi/ml⁴⁵Ca²⁺. The cells were rinsed immediately three times with PSS without⁴⁵Ca²⁺, and the amount of⁴⁵Ca²⁺ in the cells was determined by liquid scintillation spectrometry; n = 5 wells/group. B, C, Chromaffin cells were transfected with plasmids for GFP and either pCDR7 or pCMVneo, with GFP serving as a marker for the transfected cells. Cultures were loaded with 1 μm fura-2 AM and subsequently were perfused with a 10 μm concentration of a nicotinic agonist (dimethylphenylpiperazinium, DMPP), followed by the indicated concentrations of α-Ltx in PSS. The duration of each agonist application is indicated by a horizontal bar. Intracellular Ca²⁺ levels were obtained as described in Materials and Methods. Following are the mean resting Ca²⁺ levels: −CIRL, 56 ± 11 nm,n = 3; +CIRL, 45 ± 7 nm,n = 4.

Fig. 5.

Expression of CIRL increases the sensitivity of permeabilized chromaffin cells to the secretion-enhancing effects of α-Ltx. A, Monolayer cultures of nontransfected chromaffin cells were labeled with ³H-norepinephrine (³H-NE), rinsed, and incubated with the indicated concentrations of α-Ltx in physiological saline without Ca²⁺ or Mg²⁺ and with 0.2 mm EGTA for 4 min. The toxin was removed, and the cells were permeabilized for 6 min in KGEP buffer containing 20 μm digitonin and 2 mm MgATP, followed by incubation with or without 30 μm Ca²⁺in KGEP with 2 mm MgATP for 16 min. The incubation solution was removed, the cells were lysed with 1% Triton X-100, and the amount of ³H-NE was determined by liquid scintillation spectrometry. B, C, Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7 or pCMVneo, as in Figure 2. After 4–6 d the cells were incubated with the indicated concentrations of α-Ltx in PSS without Ca²⁺ or Mg²⁺ and with 0.1 mm EGTA. After 4 min the toxin was removed, and the cells were permeabilized with 20 μm digitonin in KGEP buffer without Ca²⁺ for 4 min, followed by incubation with or without 30 μm Ca²⁺ in KGEP for 15 min. MgATP (2 mm) was included in both incubations. The amounts of hGH (B) and catecholamine (C) released into the medium and the amounts remaining in the cells were determined as described. For each condition (± CIRL), release in the presence of α-Ltx was normalized to release in the absence of toxin. Actual values for Ca²⁺-dependent release of hGH in the absence of α-Ltx were 21.38 ± 0.80% (pCMVneo) and 15.75 ± 1.78% (pCDR7;p < 0.05 vs pCMVneo). Values for Ca²⁺-dependent release of catecholamine were 17.15 ± 0.59% (pCMVneo) and 17.49 ± 0.72% (pCDR7); n = 4 wells/group.

Fig. 6.

Expression of CIRL reduces secretion at all Ca²⁺ concentrations. Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7 or pCMVneo, as in Figure 2. After 4 d the cells were permeabilized with 20 μm digitonin in KGENP buffer (potassium glutamate, EGTA, nitrilotriacetic acid, and PIPES-containing solution; see Materials and Methods) without Ca²⁺ for 4 min, followed by incubation with the indicated Ca²⁺concentrations in KGENP for 15 min. MgATP (2 mm) was included in both incubations. The amounts of hGH (A) and catecholamine (B) released into the medium and the amounts remaining in the cells were determined as described; n = 4 wells/group. *p < 0.05 versus pCMVneo; **p< 0.001 versus pCMVneo; ***p < 0.0001 versus pCMVneo.

Fig. 7.

Time course of secretion in permeabilized chromaffin cells with or without overexpressed CIRL. Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7 or pCMVneo, as in Figure 2. After 4 d the cells were permeabilized with 20 μm digitonin in KGEP buffer without Ca²⁺ and with 2 mm MgATP for 4 min, followed by incubation with or without 30 μmCa²⁺ in KGEP containing 2 mm MgATP for the indicated times. The amounts of hGH (A) and catecholamine (B) released into the medium and the amounts remaining in the cells were determined as described;n = 4 wells/group.

Fig. 8.

Effect of overexpressed CIRL on ATP-dependent and ATP-independent secretion. Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7 or pCMVneo, as in Figure 2. After 4 d the cells were permeabilized with 20 μm digitonin in KGEP buffer without Ca²⁺ and with or without 2 mm MgATP for 4 min, followed by incubation with or without 30 μmCa²⁺ in the continuing presence or absence of 2 mm MgATP for 2 min (A, B) or for 2 and 15 min (C, a separate experiment). The amounts of hGH (A, C) and catecholamine (B) released into the medium and the amounts remaining in the cells were determined as described;n = 4 wells/group.

Fig. 9.

Effect of overexpressed CIRL on secretion stimulated in the presence of digitonin with and without exogenous ATP. Chromaffin cells were transfected with plasmids for hGH (pXGH5) and either pCDR7 or pCMVneo, as in Figure 2. After 6 d the cells were permeabilized with 20 μm digitonin in KGEP buffer with or without 30 μm Ca²⁺ and with or without 2 mm MgATP for 5 min. The amounts of hGH (A) and catecholamine (B) released into the medium and the amounts remaining in the cells were determined as described; n = 4 wells/group.

Fig. 10.

Effect of overexpressed G-protein-coupled receptors on secretion from digitonin-permeabilized chromaffin cells. Chromaffin cells were transfected with plasmids for hGH (pXGH5) and the α₂ adrenergic receptor, the bombesin receptor, or pCMVneo, as in Figure 2. After 4 d the cells were permeabilized (A) with 20 μm digitonin in KGEP buffer without Ca²⁺ and with 2 mm MgATP for 4 min, followed by incubation with or without 30 μmCa²⁺ in KGEP containing 2 mm MgATP for 2 min. B, Intact cells transfected with either pCMVneo or bombesin receptor were challenged for 12 min with 20 nmbombesin in PSS containing 2.2 mm Ca²⁺and 0.5 mm Mg²⁺. The amounts of hGH and catecholamine (data not shown) released into the medium and the amounts remaining in the cells were determined as described;n = 4 wells/group.