Alpha-dendrotoxin acceptor from bovine brain is a K+ channel protein. Evidence from the N-terminal sequence of its larger subunit

High affinity acceptors for alpha-dendrotoxin, a selective probe for certain fast activating voltage-dependent K+ channels, were purified approximately 4,000-fold from synaptic plasma membranes of bovine cerebral cortex. Although the preparation possessed a low content of high affinity sites for beta-bungarotoxin, antagonism of alpha-dendrotoxin binding by the latter required high concentrations; this indicates that more than one acceptor subtype has been purified. After deglycosylation of the acceptor, the sizes of its subunits were determined electrophoretically to be Mr 65,000 and Mr 39,000. Solid phase microsequencing of these isolated subunits showed that the smaller one had a blocked N terminus, but the Mr 65,000 protein gave a sequence of 27 residues. This is virtually identical to the N-terminal sequence deduced from cDNA of RCK 5, a K+ channel protein from rat brain known to be susceptible to alpha-dendrotoxin. This first report on the partial sequence of any K+ channel protein confirms that the extensive information acquired to date on the alpha-dendrotoxin acceptors is pertinent to functional neuronal K+ channels.     

Inhibition of chloroplast ATPase by the K+ channel blocker alpha-dendrotoxin.

Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high-affinity toxins mainly targeted against voltage-sensitive K+ channels. In particular, the effect of the K(+)-channel blocker alpha-dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes was characterised. Alpha-dendrotoxin (K(i) approximately 5.05 microM) and the LQ venom (K(i) approximately 1.55 micrograms/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes. Our results show that alpha-dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca(2+)-dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 microM alpha-dendrotoxin, while the crude LQ venom, at concentrations up to 10 micrograms/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by alpha-dendrotoxin indicates that approximately 2 mol alpha-dendrotoxin bind/mol CF0CF1 and 1 mol alpha-dendrotoxin/mol CF1. Known inhibitors of H(+)-flow-through CF0 acted in the presence of alpha-dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H(+)-flow-through CF0, increased the leak conductance through CF0 in the presence of alpha-dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CF0, but induces a conformation which closes the channel part for H+. Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767-771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28-Leu53) to the helix 5 and helix 6 (S5-S6) linker region (Ala413-Cys462; the charybdotoxin-binding site) of the shaker K+ channel. According to secondary-structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane alpha-helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.     

Purinergic Modulation of Hippocampal Acetylcholine Release Involves α-Dendrotoxin-Sensitive Potassium Channels

Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 microM tetrodotoxin, but it could be inhibited in a dose-dependent manner by a muscarinic agonist (10-100 nM oxotremorine) and a purinergic agonist (10-100 nM 2-chloroadenosine). The alpha-dendrotoxin (100 nM), which selectively blocks voltage-gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, alpha-dendrotoxin reduced the 2-chloroadenosine-induced inhibition of release, but did not alter the oxotremorine-induced inhibition. These results suggest that an alpha-dendrotoxin-sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.     

Electrophysiological characterization of a new member of the RCK family of rat brain K+ channels

A novel member of the RCK family of rat brain K+ channels, called RCK2, has been sequenced and expressed in Xenopus oocytes. The K+ currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC50 = 3.3 microM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329-3333] but relatively sensitive to 4-aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), alpha-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K+ channel with interesting pharmacological properties.     

Oligomeric properties of alpha-dendrotoxin-sensitive potassium ion channels purified from bovine brain.

Neuronal acceptors for alpha-dendrotoxin (alpha-DTX) have recently been purified from mammalian brain and shown to consist of two classes of subunit, a larger (approximately 78,000 M(r)) protein (alpha) whose N-terminal sequence is identical to that of a cloned, alpha-DTX-sensitive K+ channel, and a novel M(r) 39,000 (beta) polypeptide of unknown function. However, little information is available regarding the oligomeric composition of these native molecules. By sedimentation analysis of alpha-DTX acceptors isolated from bovine cortex, two species have been identified. A minority of these oligomers contain only the larger protein, while the vast majority possess both subunits. Based on accurate determination of the molecular weights of these two forms it is proposed that alpha-DTX-sensitive K+ channels exist as alpha 4 beta 4 complexes because this combination gives the best fit to the experimental data.     

Characterisation of Binding Sites for δ-Dendrotoxin in Guinea-Pig Synaptosomes: Relationship to Acceptors for the K+-Channel Probe α-Dendrotoxin

With use of biologically active 125I-labelled delta-dendrotoxin, a putative K+-channel ligand, homogeneous, noninteracting, high-affinity acceptors (KD = 0.32 +/- 0.07 nM; Bmax = 0.33 +/- 0.04 pmol/mg) were observed in synaptosomes from guinea-pig cortex. This binding was antagonised noncompetitively by alpha-dendrotoxin, an inhibitor of certain fast-activating, voltage-gated K+ channels. Chemical cross-linking of the delta-dendrotoxin-acceptor complex in synaptosomes yielded two specifically labeled polypeptides with molecular masses of 69 and 82 kilodaltons. Although alpha-dendrotoxin prevents the labelling of both these bands, it cross-linked only a single protein with a molecular mass of 69 kilodaltons. It is concluded that delta-dendrotoxin interacts with a distinct site on the oligomeric acceptors for alpha-dendrotoxin.     

Hemitoxin, the first potassium channel toxin from the venom of the Iranian scorpion Hemiscorpius lepturus

Hemitoxin (HTX) is a new K+ channel blocker isolated from the venom of the Iranian scorpion Hemiscorpius lepturus. It represents only 0.1% of the venom proteins, and displaces [125 I]alpha-dendrotoxin from its site on rat brain synaptosomes with an IC50 value of 16 nm. The amino acid sequence of HTX shows that it is a 35-mer basic peptide with eight cysteine residues, sharing 29-69% sequence identity with other K+ channel toxins, especially with those of the alphaKTX6 family. A homology-based molecular model generated for HTX shows the characteristic alpha/beta-scaffold of scorpion toxins. The pairing of its disulfide bridges, deduced from MS of trypsin-digested peptide, is similar to that of classical four disulfide bridged scorpion toxins (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8). Although it shows the highest sequence similarity with maurotoxin, HTX displays different affinities for Kv1 channel subtypes. It blocks rat Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 13, 16 and 2 nM, respectively. As previous studies have shown the critical role played by the beta-sheet in Kv1.3 blockers, we suggest that Arg231 is also important for Kv1.3 versus Kv1.2 HTX positive discrimination. This article gives information on the structure-function relationships of Kv1.2 and Kv1.3 inhibitors targeting developing peptidic inhibitors for the rational design of new toxins targeting given K+ channels with high selectivity.     

Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Charybdotoxin (ChTX), a potent peptidyl inhibitor of several types of K+ channels, binds to sites in vascular smooth muscle sarcolemma (Vázquez, J., Feigenbaum, P., Katz, G. M., King, V. F., Reuben, J. P., Roy-Contancin, L., Slaughter, R. S., Kaczorowski, G. J., and Garcia, M. L. (1989) J. Biol. Chem. 265, 20902-20909) which are functionally associated with a high conductance Ca2(+)-activated K+ channel (PK,Ca). 125I-ChTX also binds specifically and reversibly to a single class of sites in plasma membranes prepared from rat brain synaptosomes. These sites exhibit a Kd of 25-30 pM, as measured by either equilibrium or kinetic binding protocols and display a maximum density of about 0.3-0.5 pmol/mg of protein. Competition studies with native ChTX yield a Ki of 8 pM for the noniodinated toxin. The highest density of ChTX sites exists in vesicle fractions of plasma membrane origin. Binding of 125I-ChTX is modulated by metal ions that interact with K+ channels: Ba2+, Ca2+, and Cs+ cause inhibition of ChTX binding; Na+ and K+ stimulate binding at low concentration before producing complete inhibition as their concentration is increased. Stimulation of binding is due to an allosteric interaction that decreases Kd whereas inhibition results from an ionic strength effect. Tetraethylammonium ion has no effect on binding, but tetrabutylammonium ion blocks binding with a Ki of 2.5 mM. Different toxins (i.e. alpha-dendrotoxin, noxiustoxin) that inhibit an inactivating, voltage-dependent K+ channel (PK,V) block 125I-ChTX binding in brain. In marked contrast, iberiotoxin, a selective inhibitor of PK,Ca, has no effect on ChTX binding in this preparation. Inhibition of ChTX binding by alpha-dendrotoxin and noxiustoxin results from an allosteric interaction between separate binding sites for these agents and the ChTX receptor. Taken together, these results suggest that the ChTX sites present in brain are associated with PK,V rather than with PK,Ca. Therefore, 125I-ChTX is a useful probe for elucidating the biochemical properties of a number of different types of K+ channels.     

Physiological role of dendrotoxin-sensitive K+ channels in the rat cerebellar Purkinje neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.     

Characterization of a chloroplast inner envelope K+ channel.

A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.     

Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K channel principal subunit

We have identified and cloned a new member of the mammalian tandem pore domain K+ channel subunit family, TWIK-originated similarity sequence, from a human testis cDNA library. The 939 bp open reading frame encodes a 313 amino acid polypeptide with a calculated Mr of 33.7 kDa. Despite the same predicted topology, there is a relatively low sequence homology between TWIK-originated similarity sequence and other members of the mammalian tandem pore domain K+ channel subunit family group. TWIK-originated similarity sequence shares a low (< 30%) identity with the other mammalian tandem pore domain K+ channel subunit family group members and the highest identity (34%) with TWIK-1 at the amino acid level. Similar low levels of sequence homology exist between all members of the mammalian tandem pore domain K+ channel subunit family. Potential glycosylation and consensus PKC sites are present. Northern analysis revealed species and tissue-specific expression patterns. Expression of TWIK-originated similarity sequence is restricted to human pancreas, placenta and heart, while in the mouse, TWIK-originated similarity sequence is expressed in the liver. No functional currents were observed in Xenopus laevis oocytes or HEK293T cells, suggesting that TWIK-originated similarity sequence may be targeted to locations other than the plasma membrane or that TWIK-originated similarity sequence may represent a novel regulatory mammalian tandem pore domain K+ channel subunit family subunit.     

Potassium channel of cardiac sarcoplasmic reticulum is a multi-ion channel

We have characterized mechanisms of ionic permeation in the K channel of canine cardiac sarcoplasmic reticulum (SR K channel). Ionic selectivity, as measured by relative permeabilities, followed Eisenman sequence l, a low field strength sequence. Slope conductance measured in symmetrical solutions across the bilayer followed Eisenman sequence V. In all cases, the selectivity characteristics of the prominent subconductance state (O1) were similar to those of the main-state (O2). Further, our studies have revealed that this channel differs in three significant ways from the highly characterized SR K channel of skeletal muscle. First, the ratio of permeabilities Cs+ to K+ was a complex function of ion concentration. Second, the concentration dependence of conductance was not well described by the Michaelis-Menten formalism. Instead, we modeled the observed relations using a more general approach based on classical rate theory. Third, mole fraction experiments (Cs+ with K+) demonstrated a prominent anomalous effect. Certain of our Cs+ data required the Eyring rate theory approach for adequate interpretation. We adopted a symmetrical energy profile incorporating ion-ion interaction and thereby accounted for much of the data. We conclude that the canine cardiac SR K channel is significantly different from that of skeletal muscle, and it may accommodate more than one ion at a time.     

Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom.

A peptidyl inhibitor of the high conductance Ca(2+)-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus. The peptide has been named kaliotoxin (KTX). It is a single 4-kDa polypeptide chain. Its complete amino acid sequence has been determined. KTX displays sequence homology with other scorpion-derived inhibitors of Ca(2+)-activated or voltage-gated K+ channels: 44% homology with charybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% with iberiotoxin (IbTX). Electrophysiological experiments performed in identified nerve cells from the mollusc Helix pomatia showed that KTX specifically suppressed the whole cell Ca(2+)-activated K+ current. KTX had no detectable effects on voltage-gated K+ current (delayed rectifier and fast transient A current) or on L-type Ca2+ currents. KTX interacts in a one-to-one way with KCa channels with a Kd of 20 nM. Single channel experiments were performed on high conductance KCa channels excised from the above Helix neurons and from rabbit coeliac ganglia sympathetic neurons. KTX acted exclusively at the outer face of the channel. KTX applied on excised outside-out KCa channels induced a transient period of fast-flicker block followed by a persistent channel blockade. The KTX-induced block was not voltage-dependent which suggests differences in the blockade of KCa channels by KTX and by CTX. Comparison of KTX and CTX sequences leads to the identification of a short amino acid sequence (26-33) which may be implicated in the toxin-channel interaction. KTX therefore appears to be a useful tool for elucidating the molecular pharmacology of the high conductance Ca(2+)-activated K+ channel.     

Characterization of a mammalian cDNA for an inactivating voltage-sensitive K+ channel.

A cDNA clone encoding a K+ channel polypeptide with 72% amino acid sequence identity to Drosophila Shal was isolated from rat hippocampus. Functional expression of the cDNA in Xenopus oocytes generated 4-amino-pyridine-sensitive K+ channels displaying rapid inactivation kinetics. The fastest component of inactivation was slowed by the deletion of 3 basic residues in the amino-terminal region. Northern blots revealed that the mRNA encoding this K+ channel polypeptide was expressed at a similar level in the brain and in the heart. In situ hybridization revealed that the mRNA encoding this K+ channel appeared concentrated in the hippocampus, dentate gyrus, and habenular nucleus in the brain. Thus, this K+ channel polypeptide is likely to form some of the A-type K+ channels expressed in the mammalian nervous system and heart.     

Mapping the receptor site for charybdotoxin, a pore-blocking potassium channel inhibitor.

The Shaker K+ channel belongs to a family of structurally related voltage-activated cation channels that play a central role in cellular electrical signaling. By studying multiple site-directed mutants of the Shaker K+ channel, a region that forms the binding site for a pore-blocking scorpion toxin has been identified. The region contains a sequence that is highly conserved among cloned K+ channels and may contribute to the formation of the ion conduction pore.     

Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus.

An inhibitor of the high conductance, Ca2(+)-activated K+ channel (PK,Ca) has been purified to homogeneity from venom of the scorpion Buthus tamulus by a combination of ion exchange and reversed-phase chromatography. This peptide, which has been named iberiotoxin (IbTX), is one of two minor components of the crude venom which blocks PK,Ca. IbTX consists of a single 4.3-kDa polypeptide chain, as determined by polyacrylamide gel electrophoresis, analysis of amino acid composition, and Edman degradation. Its complete amino acid sequence has been defined. IbTX displays 68% sequence homology with charybdotoxin (ChTX), another scorpion-derived peptidyl inhibitor of PK,Ca, and, like this latter toxin, its amino terminus contains a pyroglutamic acid residue. However, IbTX possesses 4 more acidic and 1 less basic amino acid residue than does ChTX, making this toxin much less positively charged than the other peptide. In single channel recordings, IbTX reversibly blocks PK,Ca in excised membrane patches from bovine aortic smooth muscle. It acts exclusively at the outer face of the channel and functions with an IC50 of about 250 pM. Block of channel activity appears distinct from that of ChTX since IbTX decreases both the probability of channel opening as well as the channel mean open time. IbTX is a selective inhibitor of PK,Ca; it does not block other types of voltage-dependent ion channels, especially other types of K+ channels that are sensitive to inhibition by ChTX. IbTX is a partial inhibitor of 125I-ChTX binding in bovine aortic sarcolemmal membrane vesicles (Ki = 250 pM). The maximal extent of inhibition that occurs is modulated by K+, decreasing as K+ concentration is raised, but K+ does not affect the absolute inhibitory potency of IbTX. A Scatchard analysis indicates that IbTX functions as a noncompetitive inhibitor of ChTX binding. Taken together, these data suggest that IbTX interacts at a distinct site on the channel and modulates ChTX binding by an allosteric mechanism. Therefore, IbTX defines a new class of peptidyl inhibitor of PK,Ca with unique properties that make it useful for investigating the characteristics of this channel in target tissues.     

Permeation, selectivity, and blockade of the Ca2+-activated potassium channel of the guinea pig taenia coli myocyte

The permeation properties of the 147-pS Ca2+-activated K+ channel of the taenia coli myocytes are similar to those of the delayed rectifier channel in other excitable membranes. It has a selectivity sequence of K+ 1.0 greater than Rb+ 0.65 greater than NH4+ 0.50. Na+, Cs+, Li+, and TEA+ (tetraethylammonium) are impermeant. Internal Na+ blocks K+ channel in a strongly voltage-dependent manner with an equivalent valence (zd) of 1.20. Blockade by internal Cs+ and TEA+ is less voltage dependent, with d of 0.61 and 0.13, and half-blockage concentrations of 88 and 31 mM, respectively. External TEA+ is about 100 times more effective in blocking the K+ channel. All these findings suggest that the 147-pS Ca2+-activated K+ channel in the taenia myocytes, which functions physiologically like the delayed rectifier, is the single-channel basis of the repolarizing current in an action potential.