Venoms of elapid and hydrophid snakes contain a family of small toxic proteins called curarimimetic toxins or ␣-neurotoxins that bind with high affinity to muscular nicotinic acetylcholine receptors (AChRs) 1 and hence affect synaptic transmission (1, 2). All these toxins adopt a leaf-like shape with three adjacent loops rich in ␤-sheet that emerge from a small globular core where four disulfide bonds are invariably located (3-7). Notwithstanding their common fold and their similar biological function, ␣-neurotoxins are currently classified as short chain toxins with 60 -62 residues and four disulfide bonds and long chain toxins with 66 -74 residues and five disulfide bonds. In agreement with this old chemically based classification, we recently showed that the long chain toxins are also and uniquely capable of binding with high affinity to the neuronal ␣7 receptor (8). These preliminary data also indicated that the neuron-specific binding capacity may be associated with the unique presence in the long chain toxins of a small cyclic loop at the tip of their central loop. The goal of this work was therefore to identify as precisely as possible the determinants by which long chain toxins bind to the neuronal ␣7-AChR and to compare them with those involved when toxins bind to the muscular AChR.The toxin used in this study is ␣-cobratoxin (␣-Cbtx) (9) from Naja naja siamensis (probably Naja kaouthia (10)). It is a prototype of long chain curarimimetic toxins with a single polypeptide chain of 71 amino acids and five disulfide bonds. ␣-Cbtx binds with high affinity to the muscular type AChR from Torpedo marmorata (K d ϭ 58 pM) and the neuronal ␣7-AChR (K d ϭ 9 nM). Its three-dimensional structure is known from both NMR (11) and x-ray crystallographic studies (12). We recently submitted this toxin to an extensive site-directed mutagenesis and found that the residues by which it binds to the Torpedo AChR include a number of amino acids that are highly conserved throughout the family of curarimimetic toxins (13). These are Lys-23, Trp-25, Asp-27, Phe-29, and Arg-33, which belong to the concave face of the toxin loop II, and Lys-49, which belongs to the same face of loop III. The same residues of a short chain curarimimetic toxin are involved in binding to the same AChR (14,15). In addition, however, long and short chain curarimimetic toxins use specific residues for binding to the Torpedo AChR. These specific residues are located in the Cterminal tail and in loop I of long and short chain toxins, respectively (13).The goal of this study was 3-fold. First, using a set of 36 toxin mutants, we identified the residues by which ␣-Cbtx most likely binds to the neuronal ␣7 receptor. Second, we compared these data with those that previously indicated the residues by which the same toxin binds to the Torpedo AChR (13). Third, to identify the regions of the ␣7 receptor that are recognized by the toxin, we mutated different residues in various functional loops of the ␣7 receptor and studied the effect of these muta-* The costs of ...

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We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH. Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG. Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.z 1998 Federation of European Biochemical Societies.