A B S T R A C T Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a manner dependent upon the membrane potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external membrane surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. The K current in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a manner dependent upon the duration and interval of pulses. The recovery from block during a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. The time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2-AP interacts with the K channel more rapidly than 3-AP and 4-AP. The more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequencydependent block which is different from that in 3-AP or 4-AP. The experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley ntype gating units. Rather, the data are consistent with a simple binding scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules binding to closed K channels and being released from open channels in a voltage-dependent manner.

The group-specific protein reagents, N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an allor-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur-containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.

The effects of aminopyridines on ionic conductances of the squid giant axon membrane were examined using voltage clamp and internal perfusion techniques. 4-Aminopyridine (4-AP) reduced potassium currents, but had no effect upon transient sodium currents. The block of potassium channels by 4-AP was substantially less with (a) strong depolarization to positive membrane potentials, (b) increasing the duration of a given depolarizing step, and (c) increasing the frequency of step depolarizations. Experiments with high external potassium concentrations revealed that the effect of 4-AP was independent of the direction of potassium ion movement. Both 3- and 2-aminopyridine were indistinguishable from 4-AP except in potency. It is concluded that aminopyrimidines may be used as tools to block the potassium conductance in excitable membranes, but only within certain specific voltage and frequency limits.

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The effects of ouabain, ATP, and vanadate on palytoxin induction of ion channels were examined with the aim of elucidating the role of Na,K-ATPase in palytoxin action. Palytoxin-induced membrane depolarization of crayfish giant axons and single channel currents of frog erythrocytes and mouse neuroblastoma N1E-115 cells were examined using the intracellular microelectrode and patch-clamp techniques. External application of palytoxin in nanomolar concentrations induced depolarization in the crayfish giant axons, and the depolarization was inhibited by pretreatment of the axon with ouabain (10 microM). Internally perfused axons were less sensitive to palytoxin unless ATP (6 mM) was added internally. In patch-clamp experiments, picomolar palytoxin in the patch electrode induced single channels in both cell-attached and inside-out patches of erythrocytes and neuroblastoma cells. The induced channels had a conductance of about 10 pS, reversed near 0 mV in physiological saline solution, and was permeable to Na+, K+, Cs+, and NH4+, but not to choline. Single channel activities induced by palytoxin were inhibited by ouabain (10 microM) and vanadate (1 mM), but promoted by ATP (1 mM). The modulating effects of ouabain, vanadate, and ATP on palytoxin action suggest that the Na,K-ATPase is involved in the induction of single channels by palytoxin. Palytoxin-induced and ouabain-inhibitable single channels were observed in planar lipid bilayer incorporated with purified Na,K-ATPase. The results indicate that an interaction between palytoxin and Na,K-ATPase leads to opening of a 10-pS ion channel. They further raise the possibility that a channel structure may exist in the sodium pump which is uncovered by the action of palytoxin.

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INTRODUCTIONIn the past few years we have seen a rapid advance in the elucidation of the mechanism of action of naturally occurring neurotoxins. The time span from the first isolation of a toxin to the understanding of its mechanism of action has been shortened more and more with each newly discovered toxin. This quickening pace has been driven by a widespread interest in the potent toxicants produced by marine organisms and aided by the application of highly sophisticated biochemical and pharmacological techniques. This re view covers research on several marine neurotoxins with special reference to their recently discovered novel pharmacological actions. Some of the subjects in this area have already been reviewed. A review article by Narahashi (1) discussed the uses of chemicals as neurophysiological tools, including those of tetrodotoxin (TTX) and saxitoxin (STX). Similar subjects were later reviewed by Ritchie (2) and Catterall (3), and more recently by Pappone & Cahalan (4). Krebs (5) reviewed the recent developments in the field of marine natural products, giving comprehensive coverage of biologically ac tive compounds. Baden (6) thoroughly reviewed the natural history of marine food-borne dinoflagellate toxins. Sea anemone toxins, brevetoxins, ciguatox in, and palytoxin were discussed by Kaul & Daftari (7) in the context of bioactive substances from the sea. Ciguatoxin and maitotoxin were reviewed by Withers (8) from the medical perspective of ciguatera fish poisoning. Polypeptide neurotoxins from the sea anemone and the marine snail Conus geographus, as well as brevetoxins, were treated together with terrestrial neurotoxins in a chapter by Strichartz et al (9) on the modification of sodium channel gating. 141 0362-1642/88/0415-0141$02.00Annu. Rev. Pharmacol. Toxicol. 1988.28:141-161. Downloaded from www.annualreviews.org Access provided by Yale University -Law Library on 02/03/15. For personal use only.Quick links to online content Further ANNUAL REVIEWS 142 WU AND NARAHASHI BREVETOXINSA catastrophic episode of red tide in the Gulf of Mexico in 1946-1947 littered the beaches along the coast of Florida with tons of dead fish, with disastrous consequences to the region's economy and public health. More than 80 episodes of red tide have been recorded there since. The responsible organism was subsequently recognized as a new species of unarmored dinoflagellate and named Gymnodinium breve (10). This organism has subscquently been reclassified as Ptychodiscus brevis (11).To date, a total of eight toxins has been isolated and purified from Ptycho discus brevis. These toxins are now called brevetoxins, and a notation system, PbTx-l through PbTx-8, based on the numbering system of Shimizu (12), has been proposed to designate the various brevetoxins isolated by several labora tories (13). The brevetoxins can be divided into two subclasses according to their chemical structures: PbTx-2, -3, -5, -6 and -8 belong to one group, and PbTx-l and PbTx-7 to the other. We do not yet have information about the structure of Pb...

A B S T R A C T Deoxycholate can react with sodium channels with a high potency. The apparent dissociation constant for the saturable binding reaction is 2/zM at 8~ and the heat of reaction is ~-7 kcal/mol. Four independent tests with Na-free media, K-free media, tetrodotoxin, and pancuronium unequivocally indicate that it is the sodium channel that is affected by deoxyeholate. Upon depolarization of the membrane, the drug modified channel exhibits a slowly activating and noninactivating sodium conductance. The kinetic pattern of the modified channel was studied by increasing deoxycholate concentration, lowering the temperature, chemical elimination of sodium inactivation, or conditioning depolarization. The slow activation of the modified channel can be represented by a single exponential function with the time constant of 1-5 ms. The modified channel is inactivated only partially with a time constant of 1 s. The reversal potential is unchanged by the drug. Observations in tail currents and the voltage dependence of activation suggest that the activation gate is actually unaffected. The apparently slow activation may reflect an interaction between deoxycholate and the sodium channel in resting state.