Binding of tetrodotoxin and saxitoxin to sodium channels.

A useful first step in any chemical characterization of the sodium channels in nerve membrane would clearly be the identification of some measurable property of the channel that does not depend on the intactness of the tissue. To this end, tetrodotoxin and saxitoxin, which bind specifically to sodium channels, have been triated and their binding to rabbit, lobster and garfish non-lyelinated nerve fibres examined. In each case, a component of the binding curve was found that saturated at concentrations of a few nanomolar. In addition, non-specific binding, indicated by a linear dependence of the amount bound on concentration, occurred. A solubilized membrane preparation from garfish nerve shows the same specific binding component as that of the intact nerve. The saturable component of binding seems to reflect the sodium channel density in nerve, and this is extremely small, being about 27/mum-2 in the rabbit nerve and as small as 6/mum-2 in the garfish nerve.     

Kinetic basis for insensitivity to tetrodotoxin and saxitoxin in sodium channels of canine heart and denervated rat skeletal muscle

The single-channel blocking kinetics of tetrodotoxin (TTX), saxitoxin (STX), and several STX derivatives were measured for various Na-channel subtypes incorporated into planar lipid bilayers in the presence of batrachotoxin. The subtypes studied include Na channels from rat skeletal muscle and rat brain, which have high affinity for TTX/STX, and Na channels from denervated rat skeletal muscle and canine heart, which have about 20-60-fold lower affinity for these toxins at 22 degrees C. The equilibrium dissociation constant of toxin binding is an exponential function of voltage (e-fold per 40 mV) in the range of -60 to +60 mV. This voltage dependence is similar for all channel subtypes and toxins, indicating that this property is a conserved feature of channel function for batrachotoxin-activated channels. The decrease in binding affinity for TTX and STX in low-affinity subtypes is due to a 3-9-fold decrease in the association rate constant and a 4-8-fold increase in the dissociation rate constant. For a series of STX derivatives, the association rate constant for toxin binding is approximately an exponential function of net toxin charge in membranes of neutral lipids, implying that there is a negative surface potential due to fixed negative charges in the vicinity of the toxin receptor. The magnitude of this surface potential (-35 to -43 mV at 0.2 M NaCl) is similar for both high- and low-affinity subtypes, suggesting that the lower association rate of toxin binding to toxin-insensitive subtypes is not due to decreased surface charge but rather to a slower protein conformational step. The increased rates of toxin dissociation from insensitive subtypes can be attributed to the loss of a few specific bonding interactions in the binding site such as loss of a hydrogen bond with the N-1 hydroxyl group of neosaxitoxin, which contributes about 1 kcal/mol of intrinsic binding energy.     

A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.     

A competitive displacement assay to detect saxitoxin and tetrodotoxin

An assay is described which detects saxitoxin (STX) and tetrodotoxin (TTX) by their competitive displacement of [³H]saxitoxin from its receptor in rat brain membranes. The assay has a sensitivity of 0.15 ng STX/ml and 0.8 ng TTX/ml for buffer samples. The assay was also applied to detection of these toxins in unextracted human plasma and found to have a sensitivity of 0.5 ng STX/ml and 0.6 ng TTX/ml. The competitive displacement assay appears to be the most sensitive procedure yet for detection of STX and TTX.     

A single engineered point mutation in the adenine glycosylase MutY confers bifunctional glycosylase/AP lyase activity

The E. coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal. Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments. Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates. The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.     

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.     

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes I. Intrinsic channel and toxin parameters

The use-dependent phasic blockage of sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) was examined in frog nodes of Ranvier using trains of depolarizing pulses. The decline of the peak Na⁺ current from its initial value (I ₀) before the train to a stationary value (I ) after the train was more pronounced at more negative holding potentials. The relationship betweenI /I ₀ and holding potential was fitted by a sigmoid function which yielded values for the steepness of the voltage dependencies of around –15 mV for TTX and – 8 mV for STX. Similar values were obtained at toxin concentrations of 4 and 8 nM. The higher voltage sensitivity of STX versus TTX is interpreted in terms of the higher charge and the faster binding kinetics of STX. These differences also explain the frequency dependence of the decline of Na⁺ currents with STX (between 0.5 and 2 Hz) and the frequency independence with TTX. Variation of the pulse amplitude in a train of conditioning pulses revealed that the magnitude of the use-dependent actions of STX parallels the steady-state Na⁺ inactivation curveh . Inhibition of inactivation, by pre-treatment with chloramine-T, did not, however, abolish the use dependence. Instead, it introduced a change in the time constants of the decline of the Na⁺ currents and the magnitude became independent of the holding potential.     

Use-dependent block of sodium channels in frog myelinated nerve by tetrodotoxin and saxitoxin at negative holding potentials

Na+ currents were measured in myelinated frog nerve fibres in the presence of nanomolar concentrations of tetrodotoxin (TTX) or saxitoxin (STX) in the extracellular solution. The Na+ currents declined during a train of depolarizing pulses if the fibre was held at hyperpolarizing potentials between the pulses. At a pulse frequency of 0.8 Hz, the peak Na+ currents were reduced to 70 or 60% of the initial value in 9.3 nM TTX and 3.5 nM STX solutions, respectively. A decline of Na+ currents was also observed in two-pulse experiments. The peak Na+ current during a second test pulse did not depend on the duration (0.2 to 12 ms) of the first pulse. It decreased with increasing interval between the pulses, reached a minimum and increased again. The results are interpreted with a use-dependent blockage of Na+ channels by TTX or STX at negative holding potentials. The effects were described quantitatively, assuming a fast affinity increase of toxin receptors at Na+ channels triggered by Na+ activation followed by slow toxin binding to channels and relaxation of the receptor affinity.     

Size characteristics of the solubilized sodium channel saxitoxin binding site from mammalian sarcolemma

The sodium channel saxitoxin binding component from rat sarcolemma was solubilized with medium chain length non-ionic detergents including NP-40, Brij-96 and Lubrol-PX. Phospholipid was required for stability of the binding component. Specific saxitoxin binding was significantly temperature sensitive even with optimal levels of phospholipid present. The solubilized saxitoxin binding component chromatographed on Sepharose 6B at a position corresponding to that of a globular protein of 95--10 A Stokes radius, but had an apparent s20,w typical of a smaller molecule (s20,w = 9.2--10). Column behavior and s20,w were independent of the specific detergent used for solubilization. Anomalous column behavior may reflect molecular asymmetry, contribution from bound detergent or similar considerations.     

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes II. Extrinsic influence of cations

Use-dependent declines of Na⁺ currents in myelinated frog nerve fibres were measured during a train of depolarizing pulses in solutions containing tetrodotoxin (TTX) or saxitoxin (STX). The following effects of external monovalent (Na⁺), divalent (Ca²⁺, Mg²⁺) and trivalent (La²⁺) cations on use dependence were found: Increasing the Ca²⁺ concentration from 2 to 8 mM shifts its voltage dependence by 20 mV whereas no significant use-dependent decline occurred at 0.2 mM Ca²⁺. Doubling the external Na⁺ concentration in 0.2 mM Ca²⁺ solutions did not initiate phasic block. External Mg²⁺ ions induced a smaller, and La²⁺ ions a larger, use dependence. The time constants of the current decline were 4-fold greater in 1.08 mM La²⁺. The static block of Na⁺ currents by La³⁺ could be directly demonstrated by the relief of block during a train of pulses. The results are qualitatively explained by a toxin binding site at the Na⁺ channel whose affinity for TTX or STX depends oni) the gating conformation of the channel, probably the inactivation andii) the occupancy of a blocking site by di- or trivalent external cations.     

Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors

A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.     

A single point mutation in the novel PvCesA3 gene confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara viticola

The grapevine downy mildew, Plasmopara viticola, is one of the most devastating pathogens in viticulture. Effective control is mainly based on fungicide treatments, although resistance development in this pathogen is reported for a number of fungicides. In this study we describe for the first time the molecular mechanism of resistance to a carboxylic acid amide (CAA) fungicide. We identified a family of four cellulose synthase (CesA) genes containing conserved domains that are found in all processive glycosyltransferases. Phylogenetic analysis revealed their close relationship to the cellulose synthases of Phytophthora sp. Sequencing of the CesA genes in a CAA- resistant and -sensitive field isolate revealed five single nucleotide polymorphisms (SNPs) affecting the amino acid structure of the proteins. SNP inheritance in F₁-, F₂- and F₃-progeny confirmed resistance to be correlated with one single SNP located in PvCesA3. Only if present in both alleles, this SNP led to the substitution of a glycine for a serine residue at position 1105 (G1105S) in the deduced amino acid sequence, thus conferring CAA- resistance. Our data demonstrate that the identified genes are putative cellulose synthases and that one recessive mutation in PvCesA3 causes inheritable resistance to the CAA fungicide mandipropamid.     

A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance.

Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.     

A point mutation in domain 4-segment 6 of the skeletal muscle sodium channel produces an atypical inactivation state

We compared wild-type rat skeletal muscle NaChs (micro1) and a mutant NaCh (Y1586K) that has a single amino acid substitution, lysine (K) for tyrosine (Y), at position 1586 in the S6 transmembrane segment of domain 4. In Y1586K, macroscopic current decay is faster, the V(1/2) of the activation curve is shifted in the depolarized direction, and the fast-inactivation curve is less steep compared with mu1. After an 8-ms depolarization pulse, Y1586K recovers from inactivation much more slowly than mu1. The recovery is double exponential, suggesting recovery from two inactivation states. Varying the depolarization protocols isolates entry into an additional, "atypical" inactivation state in Y1586K that is distinct from typical fast or slow inactivation. Substitution of positively charged arginine (R) at Y1586 produces an inactivation phenotype similar to that of Y1586K. Substitution by negatively charged aspartic acid (D) or uncharged alanine (A) at Y1586 produces an inactivation phenotype similar to mu1. Our results suggest that the positive charge of lysine (K) produces the atypical inactivation state in Y1586K. We propose that a conformational change during depolarization alters the relative position of the 1586K residue in the D4-S6 segment and that atypical inactivation in Y1586K occurs via an electrostatic interaction in or near the inner pore region.     

Diversity in cardiac sodium channel disease phenotype in transgenic mice carrying a single SCN5A mutation

Lethal ventricular arrhythmias are increasingly considered an important cause of sudden death in relatively young individuals. A genetic predisposition has been recognised in many cases, and research in the last decade has focused on underlying inherited mutations in cardiac ion channels.     

A single P-loop glutamate point mutation to either lysine or arginine switches the cation-anion selectivity of the CNGA2 channel

Cyclic nucleotide-gated (CNG) channels play a critical role in olfactory and visual transduction. Site-directed mutagenesis and inside-out patch-clamp recordings were used to investigate ion permeation and selectivity in two mutant homomeric rat olfactory CNGA2 channels expressed in HEK293 cells. A single point mutation of the negatively charged pore loop (P-loop) glutamate (E342) to either a positively charged lysine or arginine resulted in functional channels, which consistently responded to cGMP, although the currents were generally extremely small. The concentration-response curve of the lysine mutant channel was very similar to that of wild-type (WT) channels, suggesting no major structural alteration to the mutant channels. Reversal potential measurements, during cytoplasmic NaCl dilutions, showed that the lysine and the arginine mutations switched the selectivity of the channel from cations (P(Cl)/P(Na) = 0.07 [WT]) to anions (P(Cl)/P(Na) = 14 [Lys] or 10 [Arg]). Relative anion permeability sequences for the two mutant channels, measured with bi-ionic substitutions, were NO(3)(-) > I(-) > Br(-) > Cl(-) > F(-) > acetate(-), the same as those obtained for anion-selective GABA and glycine channels. The mutant channels also seem to have an extremely small single-channel conductance, measured using noise analysis of about 1-2 pS, compared to a WT value of about 29 pS. The results showed that it is predominantly the charge of the E342 residue in the P-loop, rather than the pore helix dipoles, which controls the cation-anion selectivity of this channel. However, the outward rectification displayed by both mutant channels in symmetrical NaCl solutions suggests that the negative ends of the pore helix dipoles may play a role in reducing the outward movement of Cl(-) ions through these anion-selective channels. These results have potential implications for the determinants of anion-cation selectivity in the large family of P-loop-containing channels.     

The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.