Brevetoxin modulates neuronal sodium channels in two cell lines derived from rat brain.

Single Na+ channel currents were recorded from cell-attached membrane patches from two neuronal cell lines derived from rat brain, B50 and B104, and compared before and after exposure of the cells to purified brevetoxin, PbTx-3. B50 and B104 Na+ channels usually exhibited fast activation and inactivation as is typical of TTX-sensitive Na+ channels. PbTx-3 modified channel gating in both cell lines. PbTx-3 caused (1) significant increases in the frequency of channel reopening, indicating a slowing of channel inactivation, (2) a change in the voltage dependence of the channels, promoting channel opening during steady-state voltage clamp of the membrane at voltages throughout the activation range of Na+ currents, but notably near the resting potential of these cells (-60 - -50 mV), and (3) a significant, 6.7 mV hyperpolarized shift in the threshold potential for channel opening. Na+ channel slope conductance did not change in PbTx-3-exposed B50 and B104 neurons. These effects of Pbx-3 may cause hyperexcitability as well as inhibitory effects in intact brain.

Use of antimuscarinic toxins to facilitate studies of striatal m4 muscarinic receptors.

Striatal m4 muscarinic receptors are important because their blockade controls movement, and they are preferentially located on striatal neurons that project to the internal globus pallidus. The following studies were performed in vitro to provide a basis for using antimuscarinic toxins to study the effects of selective m4 blockade on movement in vivo. Because m4-toxin has limited selectivity alone (102-fold higher affinity for m4 than m1 receptors), m1-toxin was used first to occlude m1 receptors selectively, fully and irreversibly. It blocked 42% of the sites for 1.0 nM 3H-N-methylscopolamine in rat striatal membranes and 43% in sections of cat striatum. m4-Toxin (>500-fold higher affinity for m4 than m2, m3 or m5 receptors) blocked 88% of the residual, non-m1 sites in membranes, showing 64 pmol m4 receptors/g tissue. In comparison, AFDX-116, biperiden, clozapine, gallamine, hexahydrodifenidol, himbacine, R(+)hyoscyamine, methoctramine, pirenzepine, silahexocyclium, trihexyphenidyl and tripitramine did not distinguish m4 from other non-m1 receptors. 3H-Pirenzepine dissociated twice as rapidly from non-m1 as m1 receptors. Autoradiography was used to test the idea that m4 receptors are localized preferentially in the striosomes of the cat striatum. Non-m1 receptors were distributed equally in striosomes and matrix, indicating that striatal neurons with m4 receptors are in both compartments. Thus m1-toxin facilitates studies of m4 receptors by occluding m1 receptors, and m4-toxin is a selective antagonist for residual m4 receptors.

Anti-muscarinic toxins from Dendroaspis angusticeps.

Toxins from the venom of the African green mamba, Dendroaspis angusticeps, fulfill a major need for selective ligands for some of the five genetically defined subtypes of muscarinic acetylcholine receptors (m1-m5). Two toxins have been found that are highly selective antagonists for m1 and m4 receptors (m1-toxin and m4-toxin, respectively). Two other toxins (MT1 and MT2) bind with high affinity to both m1 and m4 receptors, and are agonists. Components of the venom also modify the binding of radiolabeled antagonists to m2 receptors, but an m2-selective toxin has not yet been isolated, m1-Toxin can bind to m1 receptors at the same time as typical competitive antagonists, suggesting that this toxin binds to the N-terminal and outer loops of m1 receptor molecules, rather than within the receptor pocket where typical agonists and antagonists bind. The binding of toxins to the outer parts of receptor molecules probably accounts for their much higher specificity for individual receptor subtypes than is seen with smaller ligands. Toxins are useful for identifying, counting, localizing, activating and blocking m1 and m4 receptors with high specificity.

m1-toxin.

The venom of the Eastern green mamba from Africa, Dendroaspis angusticeps, contains a number of toxins which block the binding of 3H-antagonists to genetically-defined m1 and m4 muscarinic acetylcholine receptors. Most of the anti-muscarinic activity of the venom is due to the presence of a newly-isolated toxin, "m1-toxin", which has 64 amino acids and a molecular mass of 7361 Daltons. At present m1-toxin is the only ligand which is known to be capable of fully blocking m1 receptors without affecting m2-m5 receptors. It binds very rapidly, specifically and pseudoirreversibly to the extracellular face of m1 receptors on cells, in membranes or in solution, whether or not the primary receptor site is occupied by an antagonist. Bound toxin can either prevent the binding and action of agonists or antagonists, or prevent the dissociation of antagonists. The toxin is useful for identifying m1 receptors during anatomical and functional studies, for recognizing and stabilizing receptor complexes, and for occluding m1 receptors so that other receptors are more readily studied.