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.     

Differential expression of sodium channel activities during the development of chick skeletal muscle cells in culture

The expression of Na+ channels during differentiation of cultured embryonic chick skeletal muscle cells was investigated using saxitoxin (STX) and batrachotoxin (BTX), which previously have been shown to interact with distinct, separate receptor sites of the voltage-sensitive Na+ channel of excitable cells. In the present study, parallel measurements of binding of [3H]-STX (STX) and of BTX-activated 22Na+ uptake (Na influx) were made in order to establish the temporal relationship of the appearance of these two Na+ channel activities during myogenesis. Na influx was clearly measurable in 2-d cells; from day 3 to day 7 the maximum Na influx approximately doubled when measured with saturating BTX concentrations potentiated by Leiurus scorpion toxin, while the apparent affinity of BTX, measured without scorpion toxin, also increased. Saturable STX binding did not appear consistently until day 3; from then until day 7 the STX binding capacity increased about threefold, whereas the equilibrium dissociation constant (KD) decreased about fourfold. Although Na influx in cells of all ages was totally inhibited by STX or tetrodotoxin (TTX) at 10 microM, lower concentrations (2-50 nM) blocked the influx in 7-d cells much more effectively than that in 3-d cells, where half the flux was resistant to STX at 20-50 nM. Similar but smaller differences characterized the block by TTX. In addition, when protein synthesis is inhibited by cycloheximide, both Na influx and STX binding activities disappear more rapidly in 3-d than in 7-d cells, which shows that these functions are less stable metabolically in the younger cells.     

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.     

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin- resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to - 1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.

Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.     

Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins

bTyrosine 401 of the skeletal muscle isoform (mu 1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn2+ and Cd2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na+ current for a series of point mutations at residue Tyr401 of the rat mu 1 Na channel expressed in Xenopus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na+ currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with Ki values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7- and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn2+ (Ki = 8-23 microM) required Cys, His, or Asp, three residues commonly found to coordinate directly with Zn2+ in metalloproteins. For the Cys mutant of mu 1 and also for the cardiac isoform Na channel (rh1) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na+ conductance by Zn2+ reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The Asp mutant also displayed enhanced affinity for inhibition of conductance by Ca2+ (Ki = 0.3 mM vs approximately 40 mM in wild type), but block by Ca2+ was incomplete, saturating at approximately 69% inhibition. In contrast, Cd2+ completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the particular residue at position 401 and the species of divalent cation. The His mutant also exhibited enhanced sensitivity to block by H+ with a pKa of approximately 7.5 for the His imidazole group. Our findings provide further evidence that residue 401 of mu 1 is located within the outer vestibule of the Na channel but external to the single-filing region for permeant ions.     

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.     

Tonic and phasic guanidinium toxin-block of skeletal muscle Na channels expressed in Mammalian cells

The blockage of skeletal muscle sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) have been studied in CHO cells permanently expressing rat Nav1.4 channels. Tonic and use-dependent blockage were analyzed in the framework of the ion-trapped model. The tonic affinity (26.6 nM) and the maximum affinity (7.7 nM) of TTX, as well as the "on" and "off" rate constants measured in this preparation, are in remarkably good agreement with those measured for Nav1.2 expressed in frog oocytes, indicating that the structure of the toxin receptor of Nav1.4 and Nav1.2 channels are very similar and that the expression method does not have any influence on the pore properties of the sodium channel. The higher affinity of STX for the sodium channels (tonic and maximum affinity of 1.8 nM and 0.74 nM respectively) is explained as an increase on the "on" rate constant (approximately 0.03 s(-1) nM(-1)), compared to that of TTX (approximately 0.003 s(-1) nM(-1)), while the "off" rate constant is the same for both toxins (approximately 0.02 s(-1)). Estimations of the free-energy differences of the toxin-channel interaction indicate that STX is bound in a more external position than TTX. Similarly, the comparison of the toxins free energy of binding to a ion-free, Na(+)- and Ca(2+)-occupied channel, is consistent with a binding site in the selectivity filter for Ca(2+) more external than for Na(+). This data may be useful in further attempts at sodium-channel pore modeling.     

The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field.

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) was measured at a test potential of -10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold). At Vhold of -100 mV, the ED50 was 2.1 +/- 0.4 nM, and the affinity increased to a ED50 of 1.2 +/- 0.2 nM with Vhold of -140 mV. In the absence of toxin, the peak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentials. The Woodhull model (1973) was used to describe the increase of the STX ED50 as a function of holding potential. The equivalent electrical distance of block (delta) by STX was 0.18 from the extracellular milieu when the valence of STX was fixed to +2. Analysis of the holding potential dependence of TTX block yielded a similar delta when the valence of TTX was fixed to +1. We conclude that the guanidinium toxin site is located partially within the transmembrane electric field. Previous site-directed mutagenesis studies demonstrated that an isoform-specific phenylalanine in the BrIIa channel is critical for high affinity toxin block.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Na+ channel in mammalian cardiac cells. Two kinds of tetrodotoxin receptors in rat heart membranes

The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.     

Periodate treatment reduces the tetrodotoxin-sensitivity of voltage-gated Na+ channels

(1) Voltage-clamped nerve fibres of the frog Rana esculenta were treated with periodate in the extracellular solution. (2) Periodate treatment irreversibly reduced the effect of tetrodotoxin (TTX) on the Na+ currents. (3) The effect of saxitoxin (STX) was also reduced but less than that of TTX. (4) The presence of STX during the application of periodate to the nerve fibre almost completely prevented the effect of the chemical reagent on the TTX sensitivity of the Na+ channels. (5) The reduction of the TTX effect is not due to the reaction of small amounts of periodate with the diol group of this toxin, because the effect was seen after prolonged washing with reagent-free Ringer solution with or without high amounts of ribose. (6) Carboxyl groups present in the Na+ channel seem to be quite important for the binding of TTX and STX. Periodate modifies several amino acid side chains, however, it does not attack carboxyl groups in a peptide chain. Thus, these results suggest that periodate modifies a further group critically involved in the binding of TTX and STX.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.     

Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components

The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.     

The binding of saxitoxin to axolemma of mammalian brain. Cooperative competition between saxitoxin and sodium ion.

Saturable, high affinity binding of tritium-labeled saxitoxin ([3H]STX) to axolemma-enriched membranes from white matter of bovine brain was identified. The apparent [3H]STX equilibrium dissociation constant (Kd*) was strongly affected by the cationic environment:choline ion had little effect; cesium ion increased the mammalian axolemma Kd* in a simple competitive manner. In contrast, sodium ion more dramatically increased the Kd*--this effect was highly cooperative between 75 and 200 mM sodium (Hill coefficient of 2.85). The cooperativity is most pronounced at the normally expected [sodium] external to the axon in the mammalian central nervous system. This sodium-specific cooperative modification of the STX binding site (the hypothetical "ion selectivity filter" of the axonal Na+ gate) may be indicative of some as yet undefined regulatory mechanism of the Na+ gate in mammalian myelinated axons.     

Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes.

An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.     

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.     

Insect nervous system [H]saxitoxin binding sites: characterization and target size

Saxitoxin (STX) has proved useful in the isolation and characterization of vertebrate and invertebrate voltage-operated sodium channels. Membrane extracts from the nervous system of the cockroach Periplaneta americana contain a saturable component of specific [³H]STX binding. Scatchard analysis yields a K_D of 0.84 nM, similar to that (3.0 nM) determined in electrophysiological studies on axons in the same tissue (Sattelle et al., 1979). The maximum number of binding sites, B_max (8.25 pmol/mg protein), was higher than previously observed. The specific binding component was blocked by STX and tetrodotoxin (TTX), but not by scorpion (Leiurus quinquestriatus) venom, aconitine, veratridine, sea anemone toxin and deltamethrin, which act at different sites on the channel molecule. Unlabelled STX samples prepared from different sources (Mytilus, Saxidomus and Gonyaulax) were all effective as inhibitors of [³H]STX binding. Radiation inactivation was employed to determine the molecular target size of the [³H]STX binding molecule in membranes prepared from the cockroach nervous system. By this means M_r = 171,400 ± 25,000 was estimated for the insect sodium channel.     

Biochemical characterization of plasma membrane isolated from human skeletal muscle

Specific components of ion translocation systems were studied in excitable plasma membranes isolated from normal human muscle. Na+-K+ ATPase and ouabain-sensitive K+ phosphatase activities were 8.9 +/- 1 mumol Pi/h per mg protein and 96 +/- 9 nmol/min per mg protein, respectively. Scatchard analysis of equilibrium binding assays with [3H]ouabain showed non-linear curves consistent with high- and low-affinity sites (estimated Kd 3 nM and 0.22 microM). Two families of receptors with different affinities for a tritiated TTX derivative (estimated Kd 0.4 and 4 nM) were also identified suggesting the existence in human muscle of at least two classes of voltage-dependent Na+ channels. In addition (+)-[methyl-3H]PN200-110, a potent Ca2+ antagonist used for labeling voltage-dependent Ca2+ channels, was observed to bind to a homogeneous population of receptors in the plasma membrane (Kd = 0.2 nM).     

The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin

Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX-modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX-modified channels than for open modified channels. Hence, the TTX-binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.