Insights into alpha-K toxin specificity for K+ channels revealed through mutations in noxiustoxin.

Noxiustoxin (NxTX) displays an extraordinary ability to discriminate between large conductance, calcium-activated potassium (maxi-K) channels and voltage-gated potassium (Kv1.3) channels. To identify features that contribute to this specificity, we constructed several NxTX mutants and examined their effects on whole cell current through Kv1.3 channels and on current through single maxi-K channels. Recombinant NxTX and the site-specific mutants (P10S, S14W, A25R, A25Delta) all inhibited Kv1.3 channels with Kd values of 6, 30, 0.6, 112, and 166 nM, respectively. In contrast, these same NxTX mutants had no effect on maxi-K channel activity with estimated Kd values exceeding 1 mM. To examine the role of the alpha-carbon backbone in binding specificity, we constructed four NxTX chimeras, which altered the backbone length and the alpha/beta turn. For each of these chimeras, six amino acids comprising the alpha/beta turn in iberiotoxin (IbTX) replaced the corresponding seven amino acids in NxTX (NxTX-YGSSAGA21-27-FGVDRG21-26). The chimeras differed in length of N- and C-terminal residues and in critical contact residues. In contrast to NxTX and its site-directed mutants, all of these chimeras inhibited single maxi-K channels. Under low ionic strength conditions, Kd values ranged from 0.4 to 6 microM, association rate constant values from 3 x 10(7) to 3 x 10(8) M(-1) x s(-1), and time constants for block from 5 to 20 ms. The rapid blocked times suggest that key microscopic interactions at the toxin-maxi-K channel interface may be absent. Under physiologic external ionic strength conditions, these chimera inhibited Kv1.3 channels with Kd values from 30 to 10 000 nM. These results suggest that the extraordinary specificity of NxTX for Kv1.3 over maxi-K channels is controlled, in part, by the toxin alpha-carbon backbone. These differences in the alpha-carbon backbone are likely to reflect fundamental structural differences in the external vestibules of these two channels.

Structural basis for alpha-K toxin specificity for K+ channels revealed through the solution 1H NMR structures of two noxiustoxin-iberiotoxin chimeras.

Noxiustoxin (NxTX) and iberiotoxin (IbTX) exhibit extraordinary differences in their ability to inhibit current through the large-conductance calcium-activated potassium (maxi-K) and voltage-gated potassium (Kv1.3) channels. The three-dimensional structures of NxTX and IbTX display differences in their alpha/beta turn and in the length of the alpha-carbon backbone. To understand the role of these differences in defining specificity, we constructed two NxTX mutants, NxTX-IbTX I and NxTX-IbTX II, and solved their solution structures by 1H NMR spectroscopy. For NxTX-IbTX I, seven amino acids comprising the alpha/beta turn in NxTX are replaced with six amino acids from the corresponding alpha/beta turn in IbTX (NxTX-YGSSAGA21-27FGVDRF21-26). In addition, NxTX-IbTX II contained the S14W mutation and deletion of the N- and C-terminal residues. Both NxTX-IbTX I and NxTX-IbTX II exhibit an alpha/beta scaffold structure typical of the alpha-K channel toxins. A helix is present from residues 10 to 19 in NxTX-IbTX I and from residues 13 to 19 in NxTX-IbTX II. The beta-sheet, defined by three antiparallel strands, is one residue longer in NxTX-IbTX I relative to NxTX-IbTX II. The two toxins also differ in the structure of the alpha/beta turn with NxTX-IbTX I resembling that of IbTX and with NxTX-IbTX II resembling that of NxTX. These differences in the beta-sheet and alpha/beta turn alter the dimensions of the toxin-channel interaction surface and provide insight into how these NxTX mutations alter K+ channel specificity for the maxi-K and Kv1.3 channels.

Interaction of charybdotoxin S10A with single maxi-K channels: kinetics of blockade depend on the presence of the beta 1 subunit.

The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, ChTX-S10A block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.

Electrostatic mutations in iberiotoxin as a unique tool for probing the electrostatic structure of the maxi-K channel outer vestibule.

Iberiotoxin (IbTX or alpha-KTx 1.3), a selective, high-affinity blocker of the large-conductance, calcium-activated (maxi-K) channel, exhibits a unique, asymmetric distribution of charge. To test how these charges control kinetics of IbTX binding, we generated five mutants at two positions, K27 and R34, that are highly conserved among other isotoxins. The dissociation and association rate constants, koff and kon, were determined from toxin-blocked and -unblocked durations of single maxi-K channels incorporated into planar lipid bilayers. Equilibrium dissociation constant (Kd) values were calculated from koff/kon. The IbTX mutants K27N, K27Q, and R34N caused large increases in Kd values compared to wild-type, suggesting that the IbTX interaction surface encompasses these residues. A well-established pore-blocking mechanism for IbTX predicts a voltage dependence of toxin-blocked times following occupancy of a potassium binding site in the channel pore. Time constants for block by K27R were approximately 5-fold slower at -20 mV versus +40 mV, while neutralization of K27 relieved the voltage dependence of block. This suggests that K27 in IbTX interacts with a potassium binding site in the pore. Neutralized mutants of K27 and R34, with zero net charge, displayed toxin association rate constants approximately 10-fold slower than wild-type. Association rates for R34N diminished approximately 19-fold when external potassium was increased from 30 to 300 mM. These findings suggest that simple net charge and diffusional processes do not control ingress of IbTX into the channel vestibule.

Mechanism of maxi-K channel activation by dehydrosoyasaponin-I.

Dehydrosoyasaponin-I (DHS-I) is a potent activator of high-conductance, calcium-activated potassium (maxi-K) channels. Interaction of DHS-I with maxi-K channels from bovine aortic smooth muscle was studied after incorporating single channels into planar lipid bilayers. Nanomolar amounts of intracellular DHS-I caused the appearance of discrete episodes of high channel open probability interrupted by periods of apparently normal activity. Statistical analysis of these periods revealed two clearly separable gating modes that likely reflect binding and unbinding of DHS-I. Kinetic analysis of durations of DHS-I-modified modes suggested DHS-I activates maxi-K channels through a high-order reaction. Average durations of DHS-I-modified modes increased with DHS-I concentration, and distributions of these mode durations contained two or more exponential components. In addition, dose-dependent increases in channel open probability from low initial values were high order with average Hill slopes of 2.4-2.9 under different conditions, suggesting at least three to four DHS-I molecules bind to maximally activate the channel. Changes in membrane potential over a 60-mV range appeared to have little effect on DHS-I binding. DHS-I modified calcium- and voltage-dependent channel gating. 100 nM DHS-I caused a threefold decrease in concentration of calcium required to half maximally open channels. DHS-I shifted the midpoint voltage for channel opening to more hyperpolarized potentials with a maximum shift of -105 mV. 100 nM DHS-I had a larger effect on voltage-dependent compared with calcium-dependent channel gating, suggesting DHS-I may differentiate these gating mechanisms. A model specifying four identical, noninteracting binding sites, where DHS-I binds to open conformations with 10-20-fold higher affinity than to closed conformations, explained changes in voltage-dependent gating and DHS-I-induced modes. This model of channel activation by DHS-I may provide a framework for understanding protein structures underlying maxi-K channel gating, and may provide a basis for understanding ligand activation of other ion channels.

Functional reconstitution of the large-conductance, calcium-activated potassium channel purified from bovine aortic smooth muscle.

The charybdotoxin (ChTX) receptor has been purified from bovine aortic smooth muscle using conventional chromatographic techniques and sucrose gradient centrifugation. Fractions from the final sucrose gradient purification were enriched in specific binding of monoiodinated ChTX (125i-ChTX) approximately 2000-fold over native sarcolemmal membranes. The ChTX binding activity correlated with the presence of two polypeptides of 65 (alpha) and 31 (beta) kDa. Using the cross-linking reagent, disuccinimidyl suberate, 125I-ChTX was specifically incorporated into a polypeptide of approximately 31 kDa. Cross-linking and binding of 125I-ChTX to the purified ChTX receptor was inhibited by ChTX, iberiotoxin (IbTX), and tetraethylammonium (TEA). Liposomes containing the purified ChTX receptor were incorporated into planar lipid bilayers. In symmetric 150 mM KC1, the channels observed were > 20-fold more selective for potassium over sodium and exhibited a large, single-channel conductance of 323 +/- 2.5 pS in charged lipids and 249 +/- 7 pS in neutral lipids. Depolarizing membrane potentials increased the open probability of the purified channels e-fold per 11.5 +/- 0.3 mV, while intracellular calcium increased the open probability according to a third power (2.9 +/- 0.2) relationship. Mean channel closed durations decreased while open times slightly increased as membrane potential and calcium concentration were elevated. The distributions of open and closed durations were well described by the sums of three and five to six exponential components, respectively. Purified maxi-K channels were blocked with micromolar affinity by external TEA and with nanomolar affinity by extracellular IbTX and ChTX. Kinetics of ChTX block of the purified channel revealed an equilibrium dissociation constant for toxin block 4.6 +/- 0.7 nM under conditions of physiological ionic strength. The purified maxi-K channel displays many of the biophysical and pharmacological properties of maxi-K channels derived from native tissue.

Tremorgenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels.

Tremorgenic indole alkaloids produce neurological disorders (e.g., staggers syndromes) in ruminants. The mode of action of these fungal mycotoxins is not understood but may be related to their known effects on neurotransmitter release. To determine whether these effects could be due to inhibition of K+ channels, the interaction of various indole diterpenes with high-conductance Ca(2+)-activated K+ (maxi-K) channels was examined. Paspalitrem A, paspalitrem C, aflatrem, penitrem A, and paspalinine inhibit binding of [125I]charybdotoxin (ChTX) to maxi-K channels in bovine aortic smooth muscle sarcolemmal membranes. In contrast, three structurally related compounds, paxilline, verruculogen, and paspalicine, enhanced toxin binding. As predicted from the binding studies, covalent incorporation of [125I]ChTX into the 31-kDa subunit of the maxi-K channel was blocked by compounds that inhibit [125I]ChTX binding and enhanced by compounds that stimulate [125I]ChTX binding. Modulation of [125I]ChTX binding was due to allosteric mechanisms. Despite their different effects on binding of [125I]ChTX to maxi-K channels, all compounds potently inhibited maxi-K channels in electrophysiological experiments. Other types of voltage-dependent or Ca(2+)-activated K+ channels examined were not affected. Chemical modifications of paxilline indicate a defined structure-activity relationship for channel inhibition. Paspalicine, a deshydroxy analog of paspalinine lacking tremorgenic activity, also potently blocked maxi-K channels. Taken together, these data suggest that indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date. Some of their pharmacological properties could be explained by inhibition of maxi-K channels, although tremorgenicity may be unrelated to channel block.

Purification and reconstitution of the high-conductance, calcium-activated potassium channel from tracheal smooth muscle.

The high-conductance Ca(2+)-activated K+ (maxi-K) channel from bovine tracheal smooth muscle was purified to apparent homogeneity by a combination of conventional chromatographic techniques and sucrose density gradient centrifugation. Fractions with the highest specific activity for binding of monoiodotyrosine charybdotoxin, [125I]ChTX, were enriched approximately 2000-fold over the initial digitonin-solubilized material up to a specific activity of 1 nmol/mg protein. Silver staining after SDS-polyacrylamide gel electrophoresis of the fractions from the last step of the purification indicates that binding activity is correlated with a major component of the preparation that displays an apparent molecular weight of 62,000. Labeling the same preparation with 125I-Bolton-Hunter reagent reveals the existence of both 62 (alpha)- and 31 (beta)-kDa subunits, in an apparent stoichiometry of 1:1, comigrating with binding activity. The beta subunit is heavily glycosylated. Deglycosylation studies indicate that the beta subunit represents the protein to which [125I]ChTX is covalently incorporated in the presence of the bifunctional cross-linking reagent disuccinimidyl suberate. Binding of [125I]ChTX to the purified ChTX receptor displayed the same pharmacological profile that has been found previously for toxin binding to native membranes, including inhibition by iberiotoxin, limbatustoxin, tetraethylamonium, potassium, cesium, and barium. The purified preparation was reconstituted into liposomes which were then fused with artificial lipid bilayers. Single channels were readily observed with a conductance of 235 picosiemens in 150 mM KCl that displayed selectivity for potassium over chloride and that were blocked by ChTX. The open probability of these channels was increased by depolarizing membrane potentials and by raising the internal calcium concentration. These data suggest that the maxi-K channel purified from tracheal smooth muscle is composed of two subunits.

An activator of calcium-dependent potassium channels isolated from a medicinal herb.

Large-conductance calcium-dependent potassium (maxi-K) channels play an important role in regulating the tone of airway smooth muscle and the release of bronchoconstrictive substances from nerves in the lung. Crude extracts of Desmodium adscendens, a medicinal herb used in Ghana as a treatment for asthma, inhibit binding of monoiodotyrosine charybdotoxin (125I-ChTX) to receptor sites in bovine tracheal smooth muscle membranes that have been shown to be associated with maxi-K channels. Using this assay, three active components have been purified and identified by NMR and MS. Comparison with authentic samples revealed the three active components as the known triterpenoid glycosides dehydrosoyasaponin I (DHS-I), soyasaponin I, and soyasaponin III. The most potent of these compounds, DHS-I, is a partial inhibitor of 125I-ChTX binding (Ki = 120 nM, 62% maximum inhibition). Inhibition of 125I-ChTX binding is primarily due to a decrease in the observed maximum number of binding sites, with a smaller decrease in affinity. DHS-I increases the rate of toxin dissociation from its receptor, suggesting that modulation of ChTX binding occurs through an allosteric mechanism. DHS-I reversibly increases the open probability of maxi-K channels from bovine tracheal smooth muscle incorporated into planar lipid bilayers when applied to the intracellular, but not the extracellular, side of the membrane at concentrations as low as 10 nM. In contrast, DHS-I had no effect on several other types of potassium channels or membrane transporters. This natural product is the first example of a high-affinity activator of calcium-dependent potassium channels and is the most potent known potassium channel opener.

Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels.

Charybdotoxin (ChTX) and iberiotoxin (IbTX) are highly charged peptidyl toxins which exhibit 68% sequence identity and share a similar three-dimensional structure. Despite these structural similarities, IbTX and ChTX differ in their selectivity for two types of potassium channels; large conductance calcium-activated potassium (maxi-K) channels and slowly inactivating voltage-gated (Kv1.3) potassium channels. ChTX blocks with high affinity both maxi-K and Kv1.3 channels, while IbTX blocks the maxi-K but not the voltage-gated channel. To identify regions of the toxins which impart this this selectivity, we have constructed by solid-phase synthesis two chimeric toxins, ChTX1-19IbTX20-37 (Ch-IbTX) and IbTX1-19ChTX20-37 (Ib-ChTX), as well as a truncated peptide, ChTX7-37. These peptides were assayed for their ability to inhibit [125I]ChTX binding in sarcolemmal vesicles from smooth muscle (maxi-K binding) and [125I]ChTX binding to plasma membranes from brain (Kv1.3 binding). The ability of the peptides to block the maxi-K channel was determined from recordings of single maxi-K channels incorporated into planar lipid bilayers. Block of Kv1.3 was determined from recordings of whole cell currents in Xenopus oocytes injected with mRNA encoding the cloned Kv1.3 channel. Both chimeric toxins inhibited [125I]ChTX binding to sarcolemmal membranes from smooth muscle, and they both blocked the maxi-K channel in planar lipid bilayers. In contrast, [125I]ChTX binding in brain and Kv1.3 currents expressed in oocytes were inhibited only by the chimera Ib-ChTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of iberiotoxin block of the large-conductance calcium-activated potassium channel from bovine aortic smooth muscle.

The interaction of iberiotoxin (IbTX) with the large-conductance calcium-activated potassium (maxi-K) channel was examined by measuring single-channel currents from maxi-K channels incorporated into planar lipid bilayers. Addition of nanomolar concentrations of IbTX to the external side of the channel produced long nonconducting silent periods, which were interrupted by periods of normal channel activity. The distributions of durations of blocked and unblocked periods were both described by single exponentials. The mean duration of the unblocked periods decreased in proportion with the external concentration of IbTX, while the mean duration of the blocked periods was not affected. These results suggest that IbTX blocks the maxi-K channel through a simple bimolecular binding reaction where the silent periods represent times when a single toxin molecule is bound to the channel. In symmetric solutions of 150 mM KCl, with a membrane potential of 40 mV, the mean duration of the blocked periods produced by IbTX was 840 s, and the association rate was 1.3 x 10(6) M-1 s-1, yielding an equilibrium dissociation constant of about 1 nM. Raising the internal potassium concentration increased the dissociation rate constant of IbTX in a manner which was well described by a saturable binding function for potassium. External tetraethylammonium ion increased the average duration of the unblocked periods without affecting the blocked periods, suggesting that tetraethylammonium and IbTX compete for the same site near the conductance pathway of the channel. Increasing the external concentration of monovalent cations from 25 to 300 mM with either potassium or sodium decreased the rate of binding of IbTX to the channel by approximately 24-fold, with little effect on the rate of toxin dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)

In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity.

The secondary quinone-binding site (QB site) of bacterial reaction centers from Rhodobacter sphaeroides is generally regarded to be highly specific for its native ubiquinone-10 molecule. We demonstrate here that this is a misconception rooted in the kinetic methods used to assay for occupancy of a quinone in the QB site. We show that observance of occupancy of the QB site, revealed by kinetic assay, is sensitive to the free-energy difference for electron transfer between the quinone at the primary quinone-binding site (QA site) and the QB site (-delta G0e-). For many of the compounds previously tested for binding at the QB site, the -delta G0e- between QA and QB is too small to permit detection of the functional quinone in the QB site. With an increased -delta G0e- achieved by replacing the native ubiquinone-10 at the QA site with lower-potential quinones or by testing higher-potential QB candidates, it is shown that the QB site binds and functions with the unsubstituted 1,4-benzoquinone, 1,4-naphthoquinone, and 9,10-phenanthraquinone, as well as with their various substituted forms. Moreover, quinones with the ortho-carbonyl configuration appear to function in a similar manner to quinones with the para-carbonyl configuration.

Two distinct quinone-modulated modes of antimycin-sensitive cytochrome b reduction in the cytochrome bc1 complex.

Reduction of cytochrome b-560 (analogous to cyt b-562 of mitochondria) via an antimycin-sensitive route has been revealed in chromatophores of the photosynthetic bacterium, Rhodopseudomonas sphaeroides Ga. Indeed, the results suggest that two reductive mechanisms can be operative. One is consistent with the idea that the quinol generated at the reaction center QB site enters the Q pool and, via the Qc site, equilibrates with cytochrome b-560. The other reductive mode circumvents redox equilibrium with the pool; we consider that this could result from a direct encounter of the reaction center with the bc1 complex perhaps involving a direct QB-Qc site interaction. This latter reaction is suppressed by occupancy of the Qc site, not only by antimycin but by ubiquinol and ubiquinone.