Alpha neurotoxins and their relatives: foes and friends?

In 1963, C. C. Chang and C. Y. Lee initiated a new era in the study of neurotransmitter receptors by showing that a small protein toxin derived from the Taiwanese snake Bungarus multicinctus bound tightly and specifically to the nicotinic acetylcholine receptor (nAChR) at the vertebrate neuromuscular junction (Chang and Lee 1963xChang, C.C. and Lee, C.Y. Arch. Int. Pharmacodyn. 1963; 144: 241–257PubMedSee all ReferencesChang and Lee 1963). At the time of the discovery of α-bungarotoxin (αBtx) and its cobratoxin relatives, the nAChR, although physiologically and pharmacologically well defined, was a molecular enigma. Even the question of whether it was a protein was disputed. The advent of αBtx and related α neurotoxins completely transformed the field. Their extremely high affinity and long off-times (making them essentially irreversible antagonists), their extraordinary specificity, and the ease with which radioactive or fluorescent labels are incorporated made the α neurotoxins indispensable experimental tools for the study of nAChRs. Their use immediately led to the identification, purification, and localization of these receptors in muscle and in the Torpedo electric organ, and made the nAChR the paradigmatic neurotransmitter receptor for the next 20 years.αBtx is an effective toxin because it inhibits the binding of acetylcholine to the AChR, producing neuromuscular blockade and quick death. This efficient strategy is used by a large number of poisonous snakes in the Elapidae family (cobras, kraits, mambas, and tiger snakes, among others), whose venoms collectively contain over 100 toxins with sequence homology to αBtx. All of these toxins share a common structure consisting of a β sheet core that is tightly cross-linked by four invariant disulfide bonds. Three flexible loops extend from the core to bind the receptor (Love and Stroud 1986xLove, R.A. and Stroud, R.M. Prot. Eng. 1986; 1: 37–46Crossref | PubMed | Scopus (105)See all ReferencesLove and Stroud 1986). Most of the toxins in the family bind the nAChR, but some bind other molecules, including muscarinic AChRs and acetylcholinesterase.The idea that potent toxins have evolved to recognize physiological binding sites for a natural ligand such as acetylcholine is a familiar one. Recent studies by Miwa et al. (1999), however, reported in this issue of Neuron, give this concept a new twist. They suggest that the α neurotoxins not only mimic but also may be evolutionarily related to an endogenous ligand for the AChR, one that enhances rather than inhibits the action of acetylcholine. This new finding not only sheds light on the evolutionary origins of this family of toxins, but also poses the question of how the endogenous peptide might function physiologically to modulate the AChR.The beneficent new relative of the family of elapid neurotoxins is lynx1, a small (11 kDa) protein that was identified in the course of a search for developmentally regulated genes in the cerebellum. A database search using the amino acid sequence encoded by the lynx1 gene immediately revealed homology with αBtx and with the Ly-6 family, a related group of proteins that are found on the surface of mouse lymphocytes (Gumley et al. 1995xGumley, T.P., McKenzie, I.F.C., and Sandrin, M.S. Immunol. Cell Biol. 1995; 73: 277–296Crossref | PubMedSee all ReferencesGumley et al. 1995). The Ly-6 proteins have tertiary structures that are similar to αBtx and are apparently attached to the surface membrane through a glycolipid anchor, where they participate in cell–cell and cell–substrate interactions. lynx1 shares with both αBtx and Ly-6 the highly conserved motif of eight cysteines, and models show that its predicted three-dimensional structure closely resembles the experimentally determined structures of αBtx and CD59, a member of the Ly-6 family. Moreover, the exon–intron boundaries of all three proteins are the same—a strong indication of a common evolutionary origin.What distinguishes lynx1, and makes it of unusual interest, is evidence suggesting that it has a functional relationship to AChRs in the nervous system. lynx1 is highly expressed in the brain, where it is associated with neurons in the cortex, in the hippocampus, and in the cerebellum. Interestingly, each of the sites of lynx1 localization in the brain is also a site at which the α7 neuronal AChR is expressed. The α7 AChR, a homooligomeric channel protein, is the only neuronal AChR in the mammalian brain with an appreciable affinity for αBtx (Lindstrom 1996xSee all ReferencesLindstrom 1996). (In the avian brain another subunit, α8, also binds αBtx.) In the cerebellum, lynx1 is associated with the cell bodies and proximal dendrites of Purkinje cells, which are sites of α7 localization and cholinergic innervation from mossy fibers (1xBarmack, N.H., Baughman, R.W., and Eckstein, F.P. J. Comp. Neurol. 1992; 317: 233–249Crossref | PubMedSee all References, 2xCaruncho, H.J., Guidotti, A., Lindstrom, J., Costa, E., and Pesold, C. Neuroreport. 1997; 8: 1431–1433Crossref | PubMedSee all References). This provocative colocalization prompted Miwa et al. (1999) to look for a functional interaction by expressing the α7 subunit in oocytes. They found by electrophysiological recording that addition of recombinant lynx1 increased the response of the α7 AChR to acetylcholine. These interesting findings suggest that lynx1 might have a role in modulating ACh activity in the nervous system and might do so by direct interaction with the AChR.Several questions remain, however. First, direct interaction of lynx1 with the AChR has yet to be demonstrated. Miwa et al. (1999) found that lynx1 facilitated the action of acetylcholine not only on the α7 AChR but also on the α4β2 receptor, a neuronal AChR that is not sensitive to αBtx (Lindstrom 1996xSee all ReferencesLindstrom 1996). Although lynx1 may have a different specificity for AChRs than αBtx, this finding at least raises the possibility that lynx1 affects the AChR indirectly by acting through an entirely different receptor. Direct interaction, if present, should be easily demonstrable by showing that lynx1 can block αBtx binding to the AChR, or that αBtx can block the binding of the lynx1/Fc fusion protein used to demonstrate lynx1 binding sites in cerebellar tissue sections.Second, if lynx1 interacts directly with the AChR, how does it act? There are few examples of peptides that directly modulate neurotransmitter receptor function. The effect of substance P on desensitization of the nAChR (Stallcup and Patrick 1980xStallcup, W.B. and Patrick, J. Proc. Natl. Acad. Sci. USA. 1980; 77: 634–638Crossref | PubMedSee all ReferencesStallcup and Patrick 1980) is a rare case. According to current ideas, αBtx binds at a site that is close to, but distinct from, the ACh binding site (Unwin 1993xUnwin, N. J. Mol. Biol. 1993; 229: 1101–1124Crossref | PubMed | Scopus (622)See all ReferencesUnwin 1993). Does lynx1 bind at the same site, and does it alter the binding of ACh or alter the channel properties of the AChR?Third, the location of lynx1 on the postsynaptic cell surface is unusual for a neuroeffector peptide. One possibility is that it could act as a retrograde signal to nicotinic or other receptors on the presynaptic terminal to modulate transmitter release. If true, this mechanism would be novel and interesting, as the peptide would presumably act only after proteolytic cleavage or, less likely, by direct membrane contact. Alternatively, lynx1 might play a different role at the synapse, perhaps more akin to Ly-6 in mediating adhesive or recognition functions in lymphocytes.Finally, one would like to know if lynx1 is found in muscle cells or motor neurons, and if it acts at the neuromuscular junction. A physiological role for lynx1 at this synapse would bring the α neurotoxin story full circle in a historically fitting and entirely satisfying way.