Odd behaviour of Li+ in frog skin ion transport

1. Frog skin epithelium has basolateral K+ channels that normally define the basolateral membrane potential between 80 and 100 mV. 2. The membrane mentioned also has almost silent chloride channels and a [Na+, K+, 2Cl-] cotransport, the latter probably maintains the high Cl- in the capital (also called syncytium) cells. 3. If the K+ channels are blocked by Ba2+ (or Li+) it is possible to demonstrate potential gating of the chloride channels of the basolateral membrane. 4. When the normal K+ channels are blocked, a potential-dependent K+ conductance slowly emerges. 5. If Li+ is substituted for outside Na+ the skin shows potential oscillations of about 40 mV at a frequency of about six per hour. 6. The anion channel inhibitor Indacrinone stops these oscillations. 7. The role of Cl- and K+ channels in these oscillations is discussed. 8. The transepithelial inward transport of Li+ requires the presence of Na+ and seems to be due to exchange of cellular Li+ against inside Na+ via the basolateral Na+/H+ exchanger.     

Comparative changes of levels of nitrendipine Ca2+ channels, of tetrodotoxin-sensitive Na+ channels and of ouabain-sensitive (Na+ + K+)-ATPase following denervation of rat and chick skeletal muscle

Three major ion transport systems, the nitrendipine-sensitive Ca2+ channels, the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase, have been studied in skeletal muscle from rat and chick after chronic denervation. It is shown that the situation found for the Ca2+ channel differs dramatically from that found for the Na+ channel and the (Na+ + K+)-ATPase and that regulation of the nitrendipine-sensitive Ca2+ channel in denervated muscle also differs widely from that of the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase which show a quite similar evolution.     

Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.     

Nucleotides and Mg2+ ions differentially regulate K+ channels and non-selective cation channels present in cells forming the stomatal complex

Voltage-dependent inward-rectifying (K(in)) and outward-rectifying (K(out)) K(+) channels are capable of mediating K(+) fluxes across the plasma membrane. Previous studies on guard cells or heterologously expressed K(+) channels provided evidence for the requirement of ATP to maintain K(+) channel activity. Here, the nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators. In addition to K(out) channels, these studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represents a new channel type distinguished from K(out) channels by fast activation kinetics, inhibition by elevated intracellular Mg(2+) concentration, permeability for K(+) as well as for Na(+) and insensitivity towards TEA(+). Similar observations made for guard cells from Zea mays and Vicia faba suggest a conserved regulation of channel-mediated K(+) and Na(+) transport in both cell types and species.     

In vivo analysis of a gain-of-function mutation in the Drosophila eag-encoded K+ channel

Neuronal Na+ and K+ channels elicit currents in opposing directions and thus have opposing effects on neuronal excitability. Mutations in genes encoding Na+ or K+ channels often interact genetically, leading to either phenotypic suppression or enhancement for genes with opposing or similar effects on excitability, respectively. For example, the effects of mutations in Shaker (Sh), which encodes a K+ channel subunit, are suppressed by loss-of-function mutations in the Na+ channel structural gene para, but enhanced by loss-of-function mutations in a second K+ channel encoded by eag. Here we identify two novel mutations that suppress the effects of a Sh mutation on behavior and neuronal excitability. We used recombination mapping to localize both mutations to the eag locus, and we used sequence analysis to determine that both mutations are caused by a single amino acid substitution (G297E) in the S2-S3 linker of Eag. Because these novel eag mutations confer opposite phenotypes to eag loss-of-function mutations, we suggest that eag(G297E) causes an eag gain-of-function phenotype. We hypothesize that the G297E substitution may cause premature, prolonged, or constitutive opening of the Eag channels by favoring the "unlocked" state of the channel.     

Prolactin, an activator of epithelial Na+ channel, inhibits basolateral K+ channels in adult tree frog skin

Adult amphibian skin actively transports Na+ from its apical to basolateral side while in turn, K+ is recycled through Na+, K+-ATPase and K+ channels located in the basolateral membrane. We previously found that PRL stimulates Na+ transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na+ channel (ENaC). If PRL also activates basolateral K+ channels, this activation would help to stimulate Na+ transport, too. Whether PRL does indeed stimulate basolateral K+ channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K(ATP) channel blocker, and tetrapentylammonium (TPA), a KCa channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K+ channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K+ current, suggesting that the (Na+)-H+ exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K+ channels in Hyla.     

Activity of BK(Ca) channel is modulated by membrane cholesterol content and association with Na+/K+-ATPase in human melanoma IGR39 cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.     

Dependence of mu-conotoxin block of sodium channels on ionic strength but not on the permeating [Na+]: implications for the distinctive mechanistic interactions between Na+ and K+ channel pore-blocking toxins and their molecular targets

Mu-conotoxins (mu-CTXs) are Na+ channel-blocking, 22-amino acid peptides produced by the sea snail Conus geographus. Although K+ channel pore-blocking toxins show specific interactions with permeant ions and strong dependence on the ionic strength (mu), no such dependence has been reported for mu-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of mu-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of mu and permeant ion concentration ([Na+]) on mu-CTX block of rat skeletal muscle (mu1, Nav1.4) Na+ channels. Mu-CTX sensitivity of wild-type and E758Q channels increased significantly (by approximately 20-fold) when mu was lowered by substituting external Na+ with equimolar sucrose (from 140 to 35 mm Na+); however, toxin block was unaltered (p > 0.05) when mu was maintained by replacement of [Na+] with N-methyl-d-glucamine (NMG+), suggesting that the enhanced sensitivity at low mu was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, k(on), as the primary determinant of the changes in affinity (k(on) increased 40- and 333-fold for mu-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200 mm Na+ was reduced to 50 mm). In contrast, dissociation rates changed <2-fold for the same derivatives under the same conditions. Experiments with additional mu-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity to external mu. Taken together, our findings indicate that mu-CTX block of Na+ channels depends critically on mu but not specifically on [Na+], contrasting with the known behavior of pore-blocking K+ channel toxins. These findings suggest that different degrees of ion interaction, underlying the fundamental conduction mechanisms of Na+ and K+ channels, are mirrored in ion interactions with pore-blocking toxins.     

Quinine inhibits multiple Na+ and K+ transport mechanisms in Ehrlich ascites tumor cells

The interaction of quinine with K+ and Na+ transport mechanisms has been investigated in Ehrlich ascites tumor cells. Quinine affects both Ca2+-dependent K+ channel and total K+ influx. Activation of Ca+-dependent K+ channels by propranolol is abolished by quinine (1 mM). In addition, quinine inhibits the ouabain-sensitive component of K+ influx with an apparent Ki of 0.32 +/- 0.02 mM and the furosemide-sensitive component with a Ki of 0.24 +/- 0.01 mM. Furthermore, a significant fraction (52%) of Na+ influx is inhibited by quinine. The same component is sensitive to amiloride, suggesting that it represents Na+/H+ antiport. Concomitant with the inhibition of K+ and Na+ transport, quinine stimulates ATP hydrolysis by 57%. The results suggest that quinine exerts broad, nonspecific effects on cellular mechanisms which serve to regulate cation transport in Ehrlich cells.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca(2+) channels are important structural determinants for the passage of Ca(2+) across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a 1S subunit of the skeletal L-type channel (Ca(v)1.1) to lysine virtually eliminates passage of Ca(2+) during step depolarizations. In this study, we examined the ability of this mutant Ca(v)1.1 channel (SkEIIIK) to conduct inward Na(+) current. When 150 mM Na(+) was present as the sole monovalent cation in the bath solution, dysgenic (Ca(v)1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na(+). Ca(2+) block of SkEIIIK-mediated Na(+) current was revealed by the substantial enhancement of Na(+) current amplitude after reduction of Ca(2+) in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na(+) currents through the mutant Ca(v)1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na(+) channel when Na(+) is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca(2+) permeability mediated by Ca(v) channels on physiological processes that extend beyond channel gating and permeability.     

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.     

Relief of Na+ block of Ca2+-activated K+ channels by external cations

The flickery block of single Ca2+-activated K+ channels that is produced by internally applied Na+ can be relieved by millimolar concentrations of external K+. This effect of K+ on the kinetics of Na+ block was studied by the method of amplitude distribution analysis described in the companion paper (Yellen, G., 1984b, J. Gen. Physiol., 84:157-186). It appears that K+ relieves block by increasing the exit rate of the blocking ion from the channel, not by competitively slowing its entrance rate. This suggests that a K ion that enters the channel from the outside can expel the blocking Na ion, which entered the channel from the inside. Cs+, which cannot carry current through the channel, and Rb+, which carries a reduced current through the channel, are just as effective as K+ in relieving the block by internal Na+. The kinetics of block by internal nonyltriethylammonium (C9) are unaffected by the presence of these ions in the external bathing solution.     

KappaM-conotoxin RIIIK, structural and functional novelty in a K+ channel antagonist

Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K(+) channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. kappaM-Conotoxin RIIIK (kappaM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K(+) channels. The functional and structural study presented here reveals that kappaM-conotoxin RIIIK blocks voltage-activated K(+) channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of kappaM-RIIIK in the C-terminal half is highly similar to that of mu-conotoxin GIIIA, a specific blocker of the skeletal muscle Na(+) channel Na(v)1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of kappaM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K(+) channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of kappaM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K(+) channel-targeted toxins, kappaM-RIIIK blocks voltage-gated K(+) channels with a pharmacophore that is not organized around a lysine-hydrophobic amino acid dyad motif.     

Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule

While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of approximately 100 s. We refer to this process as "ultra-slow inactivation". By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within approximately 60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain beta1 subunit along with the K1237E alpha subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in micro1 Na+ channels, may be analogous to C-type inactivation in Shaker K+ channels.     

Quaternary ammonium compounds as structural probes of single batrachotoxin-activated Na+ channels

Quaternary ammonium (QA) blockers are well-known structural probes for studying the permeation pathway of voltage-gated K+ channels. In this study we have examined the effects of a series of n-alkyl-trimethylammonium compounds (Cn-QA) on batrachotoxin (BTX)-activated Na+ channels from skeletal muscle incorporated into planar lipid bilayers. We found that these amphipathic QA compounds (Cn-QA where n = 10-18) block single Na+ channels preferentially from the internal side with equilibrium dissociation constants (KD) in the submicromolar to micromolar range. External application of amphipathic QA compounds is far less effective, by a factor of greater than 200. The block can be described by a QA molecule binding to a single site in the Na+ channel permeation pathway. QA binding affinity is dependent on transmembrane voltage with an effective valence (delta) of approximately 0.5. QA dwell times (given as mean closed times, tau c) increase as a function of n-alkyl chain length, ranging from approximately 13 ms for C10-QA to 500 ms for C18-QA at +50 mV. The results imply that there is a large hydrophobic region within the Na+ channel pore which accepts up to 18 methylene groups of the Cn-QA cation. This hydrophobic domain may be of clinical significance since it also interacts with local anesthetics such as cocaine and mepivacaine. Finally, like BTX-activated Na+ channels in bilayers, unmodified Na+ channels in GH3 cells are also susceptible to QA block. Amphipathic QA cations elicit both tonic and use-dependent inhibitions of normal Na+ currents in a manner similar to that of local anesthetic cocaine. We conclude that amphipathic QA compounds are valuable structural probes to study the permeation pathway of both normal and BTX-activated Na+ channels.