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J. Biol. Chem., Vol. 275, Issue 52, 41175-41183, December 29, 2000
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From the Pure
Latrophilin, Neurexin, and Their Signaling-deficient Mutants
Facilitate
-Latrotoxin Insertion into Membranes but Are Not Involved
in Pore Formation*
,§,,,,,
, and**
Biochemistry Department, Imperial College,
London, SW7 2AY, United Kingdom, Department of Biomedical
Sciences, University of Edinburgh, Edinburgh, EH8 9XD, United Kingdom,
and ¶ Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, Moscow, 117871, Russia
-latrotoxin is very inefficient at
forming channels/pores in artificial lipid bilayers or in the plasma
membrane of non-secretory cells. However, the toxin induces pores
efficiently in COS-7 cells transfected with the heptahelical receptor
latrophilin or the monotopic receptor neurexin. Signaling-deficient
(truncated) mutants of latrophilin and latrophilin-neurexin hybrids
also facilitate pore induction, which correlates with toxin binding
irrespective of receptor structure. This rules out the involvement of
signaling in pore formation. With any receptor, the -latrotoxin
pores are permeable to Ca2+ and small molecules
including fluorescein isothiocyanate and norepinephrine. Bound
-latrotoxin remains on the cell surface without penetrating
completely into the cytosol. Higher temperatures facilitate insertion
of the toxin into the plasma membrane, where it co-localizes with
latrophilin (under all conditions) and with neurexin (in the presence
of Ca2+). Interestingly, on subsequent removal of
Ca2+, -latrotoxin dissociates from neurexin but remains
in the membrane and continues to form pores. These receptor-independent
pores are inhibited by anti--latrotoxin antibodies. Our results
indicate that (i) -latrotoxin is a pore-forming toxin, (ii)
receptors that bind -latrotoxin facilitate its insertion into the
membrane, (iii) the receptors are not physically involved in the pore
structure, (iv) -latrotoxin pores may be independent of the
receptors, and (v) pore formation does not require -latrotoxin
interaction with other neuronal proteins.
*
This work was supported by the Wellcome Trust (a Senior
European Research Fellowship (to Y. A. U.), 058894 (to E. V. G.), and Project Grant 055498 (to R. H. A.)) and European Commission Biotechnology Program Grant B104CT965119 (to F. A. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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