BgK is a K؉ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 ؎ 1.0 Å. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K ؉ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the threedimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.Functional properties of proteins are frequently associated with a small number of important residues. For example, enzyme activities depend on a few residues that are essential for catalysis. Also, protein-protein recognition processes have been predicted (1) and recently demonstrated (2) to be energetically driven by a small proportion of the residues forming the contacting areas in protein-protein complexes, as identified by x-ray studies (3, 4). Among the proteins whose major functions require protein-protein interactions are animal toxins, which bind to various molecular targets, such as receptors or ion channels, using a small number of binding residues (5-8). As has been shown for enzymes (9), toxins with different architectures are capable of exerting similar functions (10). However, in contrast to enzymes, the molecular basis associated with the conservation of the function in structurally unrelated toxins remains unknown. In this paper, we show that two families of animal toxins with different folding patterns but a comparable capacity to bind to potassium channels include similar functional diads, composed of a critical lysine and an aromatic amino acid separated from each other by 6.6 Ϯ 1.0 Å. MATERIALS AND METHODS Synthesis of Toxin and Mutants-The amino acid sequence of BgK 1 was proposed a few years ago (11). However, chemical synthesis attempts, based on these data, systematically failed. The proposed amino acid sequence was therefore questioned, re-examined, and ultimately corrected.2 The revised amino acid sequence of BgK from Bunodosoma granulifera is: VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTC-ELC. BgK and each alanine-substituted analog were synthesized by solid phase synthesis using an Applied Biosystems model 431A peptide synthesizer, starting from 0.1 mmol of Rink-resin (4-(2Ј,4Ј-dimethoxyphenylhydroxymethylphenoxy resin; 0.48 mmol/g). A 10-fold excess (1 mmol) of Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acid was used and coupled in N-methylpyrrolidone in the presence of N,NЈ-dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The following side chain protections wer...

The cucurbituril family of drug delivery vehicles have been examined for their tissue specific toxicity using ex vivo models. Cucurbit[6]uril (CB[6]), cucurbit[7]uril (CB[7]) and the linear cucurbituril-derivative Motor2 were examined for their neuro-, myo- and cardiotoxic activity and compared with β-cyclodextrin. The protective effect of drug encapsulation by CB[7] was also examined on the platinum-based anticancer drug cisplatin. The results show that none of the cucurbiturils have statistically measurable neurotoxicity as measured using mouse sciatic nerve compound action potential. Cucurbituril myotoxicity was measured by nerve-muscle force of contraction through chemical and electrical stimulation. Motor2 was found to display no myotoxicity, whereas both CB[6] and CB[7] showed myotoxic activity via a presynaptic effect. Finally, cardiotoxicity, which was measured by changes in the rate and force of right and left atria contraction, was observed for all three cucurbiturils. Free cisplatin displays neuro-, myo- and cardiotoxic activity, consistent with the side-effects seen in the clinic. Whilst CB[7] had no effect on the level of cisplatin’s neurotoxic activity, drug encapsulation within the macrocycle had a marked reduction in both the drug’s myo- and cardiotoxic activity. Overall the results are consistent with the relative lack of toxicity displayed by these macrocycles in whole animal acute systemic toxicity studies and indicate continued potential of cucurbiturils as drug delivery vehicles for the reduction of the side effects associated with platinum-based chemotherapy.

The motor axonal variant of Guillain-Barré syndrome is associated with anti-GD1a immunoglobulin antibodies, which are believed to be the pathogenic factor. In previous studies we have demonstrated the motor terminal to be a vulnerable site. Here we show both in vivo and ex vivo, that nodes of Ranvier in intramuscular motor nerve bundles are also targeted by anti-GD1a antibody in a gradient-dependent manner, with greatest vulnerability at distal nodes. Complement deposition is associated with prominent nodal injury as monitored with electrophysiological recordings and fluorescence microscopy. Complete loss of nodal protein staining, including voltage-gated sodium channels and ankyrin G, occurs and is completely protected by both complement and calpain inhibition, although the latter provides no protection against electrophysiological dysfunction. In ex vivo motor and sensory nerve trunk preparations, antibody deposits are only observed in experimentally desheathed nerves, which are thereby rendered susceptible to complement-dependent morphological disruption, nodal protein loss and reduced electrical activity of the axon. These studies provide a detailed mechanism by which loss of axonal conduction can occur in a distal dominant pattern as observed in a proportion of patients with motor axonal Guillain-Barré syndrome, and also provide an explanation for the occurrence of rapid recovery from complete paralysis and electrophysiological in-excitability. The study also identifies therapeutic approaches in which nodal architecture can be preserved.

Miller-Fisher syndrome is an autoimmune neuropathy characterized by ataxia, areflexia and ophthalmoplegia, and in the majority of cases the presence of high titres of anti-GQ1b ganglioside antibodies. In an ex vivo model, human and mouse anti-GQ1b antibodies have been shown previously to induce a complement-dependent alpha-latrotoxin-like effect on the murine motor endplate, i.e. they bring about massive quantal release of acetylcholine and eventually block neuromuscular transmission. Using immunofluorescence microscopy with image analysis, we show here that the late stages of this electrophysiological effect temporally coincide with the loss of heavy neurofilament (200 kDa) and type III beta-tubulin immunostaining and structural breakdown of the nerve terminal, as demonstrated by electron microscopy. Ultrastructurally, axon terminals were disorganized, depleted of vesicles, and subdivided by the infiltrating processes of capping Schwann cells. These findings provide clear pathological evidence to support a role for anti-ganglioside antibodies in mediating nerve terminal injury and further advance the view that this site may be of importance as a target in some human neuropathies.

Cellular responses to hypoxia are tissue-specific and dynamic. However, the mechanisms that underlie this differential sensitivity to hypoxia are unknown. Large conductance voltage-and Caactivated K (BK) channels are important mediators of hypoxia responses in many systems. Although BK channels are ubiquitously expressed, alternative pre-mRNA splicing of the single gene encoding their pore-forming ␣-subunits provides a powerful mechanism for generating functional diversity. Here, we demonstrate that the hypoxia sensitivity of BK channel ␣-subunits is splicevariant-specific. Sensitivity to hypoxia is conferred by a highly conserved motif within an alternatively spliced cysteine-rich insert, the stress-regulated exon (STREX), within the intracellular C terminus of the channel. Hypoxic inhibition of the STREX variant is Ca-sensitive and reversible, and it rapidly follows the change in oxygen tension by means of a mechanism that is independent of redox or CO regulation. Hypoxia sensitivity was abolished by mutation of the serine (S24) residue within the STREX insert. Because STREX splice-variant expression is tissue-specific and dynamically controlled, alternative splicing of BK channels provides a mechanism to control the plasticity of cellular responses to hypoxia.alternative splicing ͉ KCNMA1 ͉ oxygen sensing M ammalian cell survival depends on the presence of oxygen. The lowering of oxygen tension (hypoxia) (whether from the disruption of blood flow, inhibition of gaseous exchange, or changes in cellular metabolism) can trigger a range of physiological responses that attempt to minimize the detrimental effects of hypoxia. Large-conductance Ca-and voltage-activated K (BK) channels have been identified as one of the key mediators of the response of the body to hypoxia. BK channels are important for the ''oxygen-sensing'' function of specialized tissues, such as the carotid body and neuroepithelia (1-3), as well as for determining cellular excitability in smooth muscle and neurons (4, 5). However, the responsiveness of native BK channels to changes in oxygen tension is as diverse as the tissues in which they are expressed, with some being completely insensitive to hypoxia (6) and others being potently inhibited by hypoxia (1-3). Also, cellular and tissue sensitivity to hypoxia are highly plastic (7-9), with adaptive responses that depend on prior and prevailing conditions, which may involve changes in BK channel expression (10), although the underlying mechanisms are essentially unknown.The pore-forming ␣-subunits of BK channels are encoded by a single gene (11), KCNMA1, which undergoes extensive alternative pre-mRNA splicing (12, 13). The ␣-subunits assemble as tetramers to form functional channels (14, 15). Distinct splicevariant mRNAs of ␣-subunits may be expressed in the same cell or differentially expressed between tissues or even neighboring cells (16,17). Dynamic modification of splice-variant mRNA expression (18, 19) allows plasticity in BK channel phenotype and cellular regulation (20)(21)(22). Functional ...