RESUMEN
Kv10.1 is a voltage-dependent K channel whose ectopic expression is associated with several human cancers. Additionally, Kv10.1 has structure-function properties which are not yet well understood. We are using drugs of clinical importance in an attempt to gain insight on the relationship between pharmacology and characteristic functional properties of this channel. Herein, we report the interaction of desethylamiodarone (desAd), the active metabolic product of the antiarrhythmic amiodarone with Kv10.1: desAd binds to both closed and open channels, with most inhibition taking place from the open state, with affinity ~ 5 times smaller than that of amiodarone. Current inhibition by desAd and amiodarone is not synergistic. Upon repolarization desAd becomes trapped in Kv10.1 and thereafter dissociates slowly from closed-and-blocked channels. The addition of the Cole-Moore shift plus desAd open-pore-block time courses yields an increasing phase on the steady-state inhibition curve (H∞) at hyperpolarized holding potentials. In contrast to amiodarone, desAd does not inhibit the Kv10.1 Cole-Moore shift, suggesting that a relevant hydrophobic interaction between amiodarone and Kv10.1 participates in the inhibition of the Cole-Moore shift, which is lost with desAd.
Asunto(s)
Amiodarona , Neoplasias , Humanos , Canales de Potasio Éter-A-Go-Go/metabolismo , Amiodarona/farmacología , Antiarrítmicos/farmacologíaRESUMEN
Kv10.1 (Eag1, or KCNH1) is a human potassium-selective channel associated with tumor development. In this work, we study the interaction of the drug dronedarone with Kv10.1. Dronedarone presents two chemical modifications aimed to lessen side effects produced by its parent molecule, the antiarrhythmic amiodarone. Hence, our observations are discussed within the framework of a previously reported interaction of amiodarone with Kv10.1. Additionally, we show new data regarding the interaction of amiodarone with the channels. We found that, unexpectedly, the effect of dronedarone on Kv10.1 differs both quantitatively and qualitatively to that of amiodarone. Among other observations, we found that dronedarone seems to be an open-pore blocker, in contrast to the reported behavior of amiodarone, which seems to inhibit from both open and closed states. Additionally, herein we provide evidence showing that, in spite of their chemical similarity, these molecules inhibit the K+ conductance by binding to non-overlapping, independent (non-allosterically related) sites. Also, we show that, while amiodarone inhibits the Cole-Moore shift, dronedarone is unable to inhibit this voltage-dependent characteristic of Kv10.1.
Asunto(s)
Dronedarona/farmacología , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Potenciales de Acción , Dronedarona/química , Canales de Potasio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Relación Estructura-ActividadRESUMEN
The ectopic overexpression of the voltage-dependent Eag1 (Kv10.1) K+ channel is associated with the cancerous phenotype in about 70% of human cancers and tumor cell lines. Recent reports showed that, compared with the canonical Shaker-related Kv family, Kv10.1 presents unique structural and functional properties. Herein, we report the interaction of the class III anti-arrhythmic compound amiodarone with Kv10.1. Using whole-cell patch clamp, we found that amiodarone inhibits Kv10.1 channel conductance with nanomolar affinity. Additionally, and interestingly, we also report that amiodarone inhibits the characteristic Cole-Moore shift of Eag1 channels. Our observations are interpreted considering the structural-functional characteristics of these channels. We conclude that amiodarone possibly binds with high affinity to the voltage sensor module, altering the gating of Kv10.1.
Asunto(s)
Amiodarona/farmacología , Canales de Potasio Éter-A-Go-Go/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Potenciales de Acción/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Células HEK293 , Humanos , Activación del Canal Iónico , Unión ProteicaRESUMEN
The original publication of this article contained multiple technical errors that occurred during its production and printing. These errors included sentences and paragraphs with parts missing. The Publisher regrets these mistakes.
RESUMEN
Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na+-channels revealed a typical ß-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other ß-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions.
Asunto(s)
Venenos de Escorpión/química , Escorpiones , Animales , Humanos , Espectrometría de Masas , México , Ratones , Péptidos/química , Picaduras de EscorpiónRESUMEN
This work reports the isolation, chemical and functional characterization of two previously unknown peptides purified from the venom of the scorpion Pandinus imperator, denominated Pi5 and Pi6. Pi5 is a classical K+-channel blocking peptide containing 33 amino acid residues with 4 disulfide bonds. It is the first member of a new subfamily, here defined by the systematic number α-KTx 24.1. Pi6 is a peptide of unknown real function, containing only two disulfide bonds and 28 amino acid residues, but showing sequence similarities to the κ-family of K-channel toxins. The systematic number assigned is κ-KTx2.9. The function of both peptides was assayed on Drosophila Shab and Shaker K+-channels, as well as four different subtypes of voltage-dependent K+-channels: hKv1.1, hKv1.2, hKv1.3 and hKv1.4. The electrophysiological assays showed that Pi5 inhibited Shaker B, hKv1.1, hKv1.2 and hKv1.3 channels with Kd = 540 nM, Kd = 92 nM and Kd = 77 nM, respectively, other studied channels were not affected. Of the channels tested only hKv1.2 and hKv1.3 were inhibited at 100 nM concentration of Pi6, the remaining current fractions were 68% and 77%, respectively. Thus, Pi5 and Pi6 are high nanomolar affinity non-selective blockers of hKv1.2 and hKv1.3 channels.
Asunto(s)
Péptidos/aislamiento & purificación , Bloqueadores de los Canales de Potasio/química , Venenos de Escorpión/química , Escorpiones , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Drosophila , Humanos , Leucocitos Mononucleares , Péptidos/química , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio , Células Sf9 , SpodopteraRESUMEN
The Shaker B K(+) conductance (G(K)) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K(+), Na(+)-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) in 0-K(+) solutions, and in the behavior of the channels in low K(+) was studied. The main findings are as follows. First, in 0-K(+) solutions, the presence of Na(+) ions is an important factor that speeds the collapse of G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.
Asunto(s)
Canales de Potasio/fisiología , Sodio/química , Animales , Baculoviridae , Línea Celular , Electrofisiología , Insectos , Iones , Potenciales de la Membrana/fisiología , Potasio/química , Potasio/farmacocinética , Canales de Potasio de la Superfamilia ShakerRESUMEN
A new peptide, Tc1, containing only 23 amino acids closely packed by three disulfide bridges was isolated from the Amazonian scorpion Tityus cambridgei. It blocks reversibly the Shaker B K(+)-channels with a K(d) of 65 nM and displaces binding of noxiustoxin to mouse brain synaptosome membranes. It is the shortest known peptide from scorpion venom that recognizes K(+)-channels and constitutes a new structural subfamily of toxin, classified as alphaKTx 13.1.
Asunto(s)
Neurotoxinas/metabolismo , Péptidos/metabolismo , Bloqueadores de los Canales de Potasio , Venenos de Escorpión/metabolismo , Escorpiones/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Ratones , Datos de Secuencia Molecular , Neurotoxinas/clasificación , Neurotoxinas/aislamiento & purificación , Péptidos/clasificación , Péptidos/aislamiento & purificación , Canales de Potasio/genética , Venenos de Escorpión/clasificación , Venenos de Escorpión/aislamiento & purificación , Canales de Potasio de la Superfamilia Shaker , Spodoptera/citología , Sinaptosomas/metabolismoRESUMEN
A novel toxin (TdK1) was purified from the venom of the scorpion Tityus discrepans, sequenced and functionally characterized. It contains 37 amino acid residues and blocks reversible the shakerB K+ channel expressed in SF9 cells with a Kd in the order of 280 nM. The proposed systematic nomenclature for this peptide is alpha-KTx4.3.
Asunto(s)
Bloqueadores de los Canales de Potasio , Canales de Potasio , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular , Peso Molecular , Venenos de Escorpión/aislamiento & purificación , Homología de Secuencia de Aminoácido , Canales de Potasio de la Superfamilia ShakerRESUMEN
Here we describe the basic features of the interaction of K+ channels with Pi1, a recently described 35 amino acid scorpion toxin, which has four disulfide bridges instead of the three commonly found in all the other known scorpion toxins. We found that: (a) Pi1 blocks ShakerB from the outside with a 1:1 stoichiometry, and a Kd of 32 nM in zero external [K+]; (b) extracellular K+, Rb+ and Cs+ but not NH4+ ions strongly impede (destabilize) the block by this toxin; interestingly (c) the destabilizing binding of K+, Rb+, and Cs+ is described by a Hill coefficient n > 1; (d) external K+ is more effective than internal K+ to reduce the block by Pi1.
Asunto(s)
Péptidos/antagonistas & inhibidores , Venenos de Escorpión/farmacología , Animales , Cationes , Línea Celular , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Péptidos/genética , Escorpiones/metabolismo , Canales de Potasio de la Superfamilia Shaker , Sodio/metabolismo , Spodoptera/citologíaRESUMEN
Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator, and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K+-channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K+ -channels (a Kv1.1 sub-family type of channel) at 100nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125I-Noxiustoxin, a well known K+-channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with Ic50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K -channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important 'point mutations' responsible for such impressive variation in blocking properties of both toxins, here described.
Asunto(s)
Péptidos/aislamiento & purificación , Péptidos/toxicidad , Bloqueadores de los Canales de Potasio , Venenos de Escorpión/química , Venenos de Escorpión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Unión Competitiva , Encéfalo/efectos de los fármacos , Caribdotoxina/química , Caribdotoxina/metabolismo , Cistina/química , Disulfuros/química , Datos de Secuencia Molecular , Péptidos/química , Ratas , Venenos de Escorpión/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Sinaptosomas/efectos de los fármacosRESUMEN
Celecoxib (Cx), an anti-inflammatory drug designed to inhibit COX2, can affect some ion channels. T-type (CaV3) channels have been implicated in sperm physiology. Here we report and characterize the Cx induced inhibition of T-type channels in mouse spermatogenic cells. Unexpectedly, Cx can also induce the acrosome reaction (AR), an intracellular Ca(2+) ([Ca(2+)]i) increase and a sperm depolarization. This [Ca(2+)]i increase possibly results from the ability Cx has to alkalinize intracellular pH (pHi), which is known to activate the sperm specific Ca(2+) channel CatSper. As the Cx induced [Ca(2+)]i increase is sensitive to mibefradil, a CatSper blocker, this channel may mediate the Cx-induced Ca(2+) entry leading to the AR. Our observations demonstrate that Cx can compromise fertilization.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Antiinflamatorios/farmacología , Canales de Calcio Tipo T/efectos de los fármacos , Pirazoles/farmacología , Espermatogénesis/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Celecoxib , Masculino , RatonesAsunto(s)
Antineoplásicos/farmacología , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Mibefradil/farmacología , Animales , Antihipertensivos/farmacología , Transporte Biológico , Canales de Potasio Éter-A-Go-Go/química , Humanos , Potasio/química , Unión Proteica , Conformación ProteicaRESUMEN
The venom from the Brazilian scorpion Tityus stigmurus was fractionated by high performance liquid chromatography (HPLC) and the corresponding components were used for molecular mass determination using electrospray ion trap mass spectrometry. One hundred distinct components were clearly assigned showing molecular masses from 216.5 to 44,800.0 Da. Fifteen new components were isolated and sequenced, four of them to completion: Tst-3 (similar to Na(+) channel specific scorpion toxins), Tst-17 (a K(+) channel blocking peptide similar to Tc1), Tst beta KTx (a peptide with identical sequence as that of TsTX-K beta toxin earlier described to exist in T. serrulatus venom) and finally a novel proline-rich peptide of unknown function. Among the eleven components partially sequenced were two enzymes: hyaluronidase and lysozyme. The first enzyme has a molecular mass of 44,800.0 Da. This enzyme showed high activity against the substrate hyaluronan in vitro. Amino acid sequence of the second enzyme showed that it is similar to other known lysozymes, with similar molecular mass and sequence to that of bona fide lysozymes reported in public protein data banks. Finally, this communication reports a correlation among HPLC retention times and molecular masses of folded scorpion toxins as well as a comparative structural and physiological analysis of components from the venom of several species of the genus Tityus.
Asunto(s)
Proteínas de Insectos/química , Bloqueadores de los Canales de Potasio/química , Proteómica , Venenos de Escorpión/química , Escorpiones , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Electrofisiología , Hialuronoglucosaminidasa/análisis , Proteínas de Insectos/farmacología , Datos de Secuencia Molecular , Peso Molecular , Muramidasa/análisis , Técnicas de Placa-Clamp , Mapeo Peptídico , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/farmacología , Canales de Potasio de la Superfamilia Shaker/efectos de los fármacos , Canales de Potasio de la Superfamilia Shaker/metabolismo , Especificidad de la Especie , Espectrometría de Masa por Ionización de Electrospray , Spodoptera/citología , Spodoptera/efectos de los fármacosRESUMEN
1. Shaker B K+ channels, expressed in the insect cell line Sf9, were studied in zero K+, Na+ or N-methyl-D-glucamine (NMG)-containing solutions. In the absence of K+ ions on both sides of the membrane, the K+ conductance collapsed with the delivery of short depolarizing pulses that activated the channels. The collapse of the conductance was fully prevented when the channels were kept closed at a holding potential of -80 mV. 2. The fall in K+ conductance had the notable characteristic of being strikingly stable. At -80 mV or more negative holding potentials, the conductance never recovered (cells observed for up to 1 h). 3. The extent of collapse of the K+ conductance depended on the number of depolarizing activating pulses applied in zero K+ solutions. For moderate to low frequencies of pulsing (1 to 0.002 Hz), the extent of the collapse did not depend on the frequency. 4. K+, Rb+, Cs+ and NH4+ added to the external Na+ solution impeded the fall in K+ conductance. 5. TEA added to the external, zero K+, Na(+)-containing solution also precluded the fall of the conductance. The protection by TEA paralleled its block of the outward K+ currents recorded with standard recording solutions. 6. The fall in K+ conductance was prevented by depolarized holding potentials. 7. The K+ conductance that was thought to be irreversibly lost at -80 mV or more negative holding potentials was fully recovered, however, after a prolonged (tens of seconds to minutes) change in the holding potential to depolarized values (above -50 mV). Full recovery could be obtained at any time after the former halt of the conductance.
Asunto(s)
Potenciales de la Membrana/fisiología , Canales de Potasio/fisiología , Potasio/fisiología , Sodio/fisiología , Animales , Línea Celular , Electrofisiología , Factores de TiempoRESUMEN
In the absence of K(+) on both sides of the membrane, delivery of standard activating pulses collapses the Shaker B K(+) conductance. Prolonged depolarizations restore the ability to conduct K(+). It has been proposed that the collapse of the conductance results from the dwelling of the channels in a stable closed (noninactivated) state (, J. Physiol. (Lond.). 499:3-15). Here it is shown that 1) Ba(2+) impedes the collapse of the K(+) conductance, protecting it from both sides of the membrane; 2) external Ba(2+) protection (K(d) = 63 microM at -80 mV) decreases slightly as the holding potential (HP) is made more negative; 3) external Ba(2+) cannot restore the previously collapsed conductance; on the other hand, 4) internal Ba(2+) (and K(+)) protection markedly decreases with hyperpolarized HPs (-80 to -120 mV), and it is not dependent on the pulse potential (0 to +60 mV). Ba(2+) is an effective K(+) substitute, inhibiting the passage of the channels into the stable nonconducting (noninactivated) mode of gating.
Asunto(s)
Canales de Potasio/metabolismo , Animales , Bario/farmacología , Fenómenos Biofísicos , Biofisica , Línea Celular , Conductividad Eléctrica , Técnicas In Vitro , Potenciales de la Membrana , Potasio/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Potasio de la Superfamilia Shaker , Soluciones , SpodopteraRESUMEN
Fast inactivation in ShakerB K channels results from pore-block caused by "ball peptides" attached to the inner part of each K channel. We have examined the question of how many functional inactivating balls are on each channel and how this number affects inactivation and recovery from inactivation. To that purpose we expressed ShakerB in the insect cell line Sf9 and gradually removed inactivation by perfusing the cell interior with the hydrolytic enzyme papain under whole cell patch clamp. Inactivation slows down as the balls are removed by an amount consistent with the presence of four balls on each channel. Recovery from inactivation has the same time course early and late in papain action; it does not depend on the number of balls remaining on the channel, consistent with the idea that reinactivation is not significant during recovery from inactivation. Our conclusion is that ShakerB has four ball peptides, each capable of causing inactivation. Statistically, the balls are identical and independent. The stability of N-type inactivation by the remaining balls is not appreciably affected by removing some of the balls from a channel.
Asunto(s)
Péptidos/química , Bloqueadores de los Canales de Potasio , Canales de Potasio/química , Animales , Fenómenos Biofísicos , Biofisica , Línea Celular , Clonación Molecular , Péptidos y Proteínas de Señalización Intracelular , Cinética , Nucleopoliedrovirus/genética , Papaína/farmacología , Péptidos/genética , Péptidos/metabolismo , Canales de Potasio/genética , Canales de Potasio de la Superfamilia Shaker , SpodopteraRESUMEN
We have studied the relation between permeation and recovery from N-type or ball-and-chain inactivation of ShakerB K channels. The channels were expressed in the insect cell line Sf9, by infection with a recombinant baculovirus, and studied under whole cell patch clamp. Recovery from inactivation occurs in two phases. The faster of the two lasts for approximately 200 ms and is followed by a slow phase that may require seconds for completion. The fast phase is enhanced by both permeant ions (K+, Rb+) and by the blocking ion Cs+, whereas the impermeant ions (Na+, Tris+, choline+) are ineffective. The relative potencies are K+ > Rb+ > Cs+ > NH4+ >> Na+ approximately choline+ approximately Tris+. Ion permeation through the channels is not essential for recovery. The results suggest that cations influence the fast phase of recovery by binding in a site with an electrical distance greater than 0.5. Recovery from fast inactivation is voltage-dependent. With Na+, choline+, or Tris+ outside, about 15% of the channels recover in the fast phase (-80 mV), and the other 85% apparently enter a second inactivated state from which recovery is very slow. Recovery in this phase is not influenced by external ions, but is speeded by hyperpolarization.
Asunto(s)
Bloqueadores de los Canales de Potasio , Animales , Baculoviridae/genética , Fenómenos Biofísicos , Biofisica , Calcio/farmacología , Cationes/farmacología , Línea Celular , Cesio/farmacología , Expresión Génica , Transporte Iónico , Cinética , Potenciales de la Membrana , Modelos Biológicos , Permeabilidad , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio de la Superfamilia Shaker , SpodopteraRESUMEN
Recently, patch-clamping of yeast protoplasts has revealed the presence of plasma membrane K+ channels (Gustin, M. C., B. Martinac, Y. Saimi, M. R. Culberston, and C. Kung. 1986. Science (Wash. DC). 233:1195-1197). In this work we show that fusion of purified plasma membranes into planar bilayers allows the study of the yeast channels. The main cationic conductances detected were of 64 and 116 pS, however, larger and smaller conductances have been observed. The two main conductances were sensitive to the K+ channels blockers tetraethylammonium (TEA+) and Ba2+. Bionic experiments indicated that both conductances were K+ selective.
Asunto(s)
Membrana Dobles de Lípidos , Canales de Potasio/fisiología , Saccharomyces cerevisiae/fisiología , Membrana Celular/fisiología , Potenciales de la Membrana , Fosfatidilcolinas , Fosfatidiletanolaminas , Fosfatidilserinas , Protoplastos/fisiologíaRESUMEN
Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.