A Ca²⁺-Independent Receptor for α-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.

Mutant α-Latrotoxin (LTXN4C) Does Not Form Pores and Causes Secretion by Receptor Stimulation

“…6D). In agreement with the previously published data (6,22), the bell-shaped secretion curve indicated that catecholamine release was mainly stimulated by Ca 2ϩ entry via toxin pores. Indeed, while exocytosis correlated with intracellular [Ca 2ϩ ] at lower [LTX WT ], excessive Ca 2ϩ influx induced by 3-5 nM LTX WT inhibited release as in Ref.…”

“…Secretion of Catecholamines from Bovine Chromaffin Cells-LTX is known to efficiently stimulate exocytosis of LDCV containing catecholamines and hormones in chromaffin and PC12 cells; this action is strictly Ca 2ϩ -dependent (6,36).…”

Protein-tyrosine Phosphatase-ς Is a Novel Member of the Functional Family of α-Latrotoxin Receptors

“…Experimental (PTP , 1-875, or CIRL; 3 g/well) and control (pCMVneo; 3 g/well) plasmids were each mixed with pXGH5 (2 g/well) prior to generating the precipitates. Previous work in the laboratory has demonstrated that this ensures that Ͼ95% of transfected cells express both hGH and the protein of interest (36,37). hGH is stored in chromaffin granules and is released concomitantly with endogenous catecholamine by various secretagogues.…”

“…hGH was measured with a luminescent assay kit from Corning Nichols Institute Diagnostics (San Juan Capistrano, CA) (37). Endogenous catecholamines were measured by spectrofluorometric assay (40).…”

“…Chromaffin cells respond to ␣-latrotoxin stimulation by robust exocytosis of catecholamines (42,37). To distinguish the secretion from only a minor portion of actually transfected cells, we used an earlier developed method based on the cotransfection of the human growth hormone cDNA followed by the quantification of the human growth hormone secretion (43).…”