Vesicle exocytosis stimulated by α-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+

Abstract

α-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca 2⍣ -dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca 2⍣ -dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca 2⍣ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca 2⍣ and is blocked by drugs depleting Ca 2⍣ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca 2⍣ -independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca 2⍣ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTXstimulated exocytosis depends upon the co-operative action of external and intracellular Ca 2⍣ involving G proteins and PLC, whereas the Ca 2⍣ -independent release is largely non-vesicular.

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

“…NRXs) may be responsible for mediating this LTX action. Although this hypothesis contradicted our previous results (5,26,27), we decided to test it using the fact that Sr 2ϩ does not support the interaction of LTX with NRX I␣ but substitutes Ca 2ϩ in LTX-evoked exocytosis (5).The objectives of the current study were (i) to reveal the …”

“…NRXs Do Not Bind LTX N4C in the Presence of Sr 2ϩ -We showed previously that Sr 2ϩ could fully replace Ca 2ϩ in supporting LTX-evoked release of [ 3 H]norepinephrine from rat synaptosomes (5). At the same time, no interaction between NRX I␣ and LTX could be detected in the presence of Sr 2ϩ .…”

“…Second, we needed to exclude the toxin's interaction with one receptor type. NRXs were chosen for two reasons: (i) they were apparent candidates to mediate the receptor-transduced LTX action, which critically required Ca 2ϩ (5,8), and (ii) toxin binding to all members of the NRX family could be totally abolished by a simple replacement of Ca 2ϩ with Sr 2ϩ (5) (Fig. 4).…”

“…While NRX I␣ binds LTX strongly, other homologs have low affinities for the toxin (22); however, all NRXs strictly require Ca 2ϩ for this interaction (22,23). LPH is a G protein-coupled receptor that binds LTX avidly under all ionic conditions (5). LPH also has two homologs (LPH 2 and 3), but these do not interact with LTX appreciably (22,24,25).…”

Activation of phospholipase D by metabotropic glutamate receptor agonists in rat cerebrocortical synaptosomes

“…To our knowledge at present, this is the ®rst report on the e ect of mGluR agonists on IP 3 levels in synaptosomes although laterotoxin has been reported to activate synaptosomal PLC but produce only a small nonsigni®cant 5% increase in IP 3 . (Davletov et al, 1998). However, the lack of detectable IP 3 elevation together with the monophasic slow DAG increase suggests that mGluR1 and mGluR5 agonists fail to stimulate PI turnover in rat cerebrocortical synaptosomes.…”