Kinetic characteristics of the excitability-inducing material channel in oxidized cholesterol and brain lipid bilayer membranes

The kinetic characteristics of the opening and closing of the excitability-inducing material (EIM) channel in oxidized cholesterol and in brain lipid bilayers are compared. The kinetics of the opening and closing of individual ion-conducting channels in bilayers doped with small amounts of EIM are determined from discrete fluctuations in ionic current. The kinetics for approach to steady-state conductance are determined for lipid bilayers containing many channels. Steady-state and kinetic characteristics for the EIM channel incorporated in brain lipid bilayers can be accounted for by the model developed for the EIM channel incorporated in oxidized cholesterol membranes. Relaxation time, calculated from rate constants of single-channel membranes or directly measured in many-channel membranes is strongly temperature dependent, and is always shorter in brain lipid membranes. Changes in temperature do not affect the interaction of the electric field and the open channel, but the open configuration of the EIM channel in brain lipid bilayers is stablized with increasing temperature. The configurational energy difference between the open and closed channel, calculated from temperature studies, is larger in brain lipid bilayers. The energy barrier which separates the two configurations of the channel is larger in oxidized cholesterol bilayers.     

Ion Transport Through Excitability-Inducing Material (EIM) Channels in Lipid Bilayer Membranes

Two different methods were used to determine the relative permeability and the voltage-dependent conductance of several different cations in excitability-inducing material (EIM)-doped lipid bilayers. In one method, the conductances of individual channels were measured for Li, Na, K, Cs, NH₄, and Ca, and in the other method biionic potentials of a membrane with many channels were measured for Li, Na, K, Cs, and Rb. The experimental results for the two methods are in agreement. The relative permeabilities are proportional to the ionic mobilities in free aqueous solution. The voltage dependence of the conductance is the same for all cations measured.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

The Nature of the Negative Resistance in Bimolecular Lipid Membranes Containing Excitability-Inducing Material

When sufficiently small amounts of excitability-inducing material (EIM) are added to a bimolecular lipid membrane, the conductance is limited to a few discrete levels and changes abruptly from one level to another. From our study of these fluctuations, we have concluded that the EIM-doped bilayer contains ion-conducting channels capable of undergoing transitions between two states of different conductance. The difference in current between the "open" and "closed" states is directly proportional to the applied membrane potential, and corresponds to a conductance of about 3 x 10^-10 ohm^-1. The fraction of the total number of channels that is open varies from unity to zero as a function of potential. The voltage-dependent opening and closing of channels explains the negative resistance observed for bimolecular lipid membranes treated with greater amounts of EIM.     

Properties of Voltage-gated Ion Channels Formed by Syringomycin E in Planar Lipid Bilayers

Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance, the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages, as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power of syringomycin E concentration suggesting that at least six monomers are required for channel formation. Received: 3 April 1995/Revised: 24 August 1995     

Kinetics of opening and closure of syringomycin E channels formed in lipid bilayers

A cyclic lipodepsipeptide, syringomycin E (SME), incorporated into planar lipid membranes forms two types of channels ("small" and "large") different in their conductance by approximately a factor of six (Biophys. J. 74:2918-2925 (1998)). We analysed the dynamics of the SME-induced transmembrane current under voltage-clamp conditions to clarify the mechanisms of formation of these channels. The voltage-dependent opening/closure of SME channels in lipid bilayers are interpreted in terms of transitions between three types of clusters including 6-7 SME molecules and some lipid molecules. The initial cluster, the precursor of the other two, was in equilibrium with SME monomer molecules at the membrane surface. The other two types of clusters (State 1 and State 2) were formed from the precursor and also during their interconversions (the consecutive-parallel mechanism of transitions). State 1 was a non-conducting state in equilibrium with small channels, which partially determined the ionic conductance of lipid bilayers modified by SME. State 2 corresponded to large SME channels, major contributors to the conductance of a bilayer. The results of the theoretical analysis based on the chemical kinetics concepts were consistent with experimental observations. Such properties of the SME-induced channels as cluster organisation, voltage dependence and the existence of a non-conducting state are all features shared by many ion channels in biological membranes. This makes it possible to use SME channels as a model to study naturally occurring ion channels.     

Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.     

Voltage-induced nonconductive pre-pores and metastable single pores in unmodified planar lipid bilayer

Electric fields promote pore formation in both biological and model membranes. We clamped unmodified planar bilayers at 150-550 mV to monitor transient single pores for a long period of time. We observed fast transitions between different conductance levels reflecting opening and closing of metastable lipid pores. Although mean lifetime of the pores was 3 +/- 0.8 ms (250 mV), some pores remained open for up to approximately 1 s. The mean amplitude of conductance fluctuations (approximately 500 pS) was independent of voltage and close for bilayers of different area (40,000 and 10 microm(2)), indicating the local nature of the conductive defects. The distribution of pore conductance was rather broad (dispersion of approximately 250 pS). Based on the conductance value and its dependence of the ion size, the radius of the average pore was estimated as approximately 1 nm. Short bursts of conductance spikes (opening and closing of pores) were often separated by periods of background conductance. Within the same burst the conductance between spikes was indistinguishable from the background. The mean time interval between spikes in the burst was much smaller than that between adjacent bursts. These data indicate that opening and closing of lipidic pores proceed through some electrically invisible (silent) pre-pores. Similar pre-pore defects and metastable conductive pores might be involved in remodeling of cell membranes in different biologically relevant processes.     

Thermodynamic and kinetic studies of the gating behavior of a K+- selective channel from the sarcoplasmic reticulum membrane

A voltage-dependent, K+-selective ionic channel from sarcoplasmic reticulum of rabbit skeletal muscle has been studied in a planar phospholipid bilayer membrane. The purpose [corrected] of this work is to study the mechanism by which the channel undergoes transitions between its conducting and nonconducting states. Thermodynamic studies show that the "open" and "closed" states of the channel exist in a voltage-dependent equilibrium, and that the channel displays only a single open state; the channel conductance is 120 pmho in 0.1 M K+. The channel's gating process follows single exponential kinetics at all voltages tested, and the individual opening and closing rate constants are exponentially dependent on voltage. The individual rate constants may also be determined from a stochastic analysis of channel fluctuations among multiple conductance levels. Neither the thermodynamic nor the kinetic parameters of gating depend on the absolute concentration of channels in the bilayer. The results are taken as evidence that the channel gates by an unusually simple two-state conformational mechanism in which the equivalent of 1.1 net charges are moved across the membrane during the formation of the open channel.     

Isolation, molecular and functional properties of the C-terminal domain of colicin A

Partial proteolytic digestion of colicin A with bromelain allowed the isolation of a 20-kd fragment. This fragment has been purified to homogeneity and its molecular properties have been studied. The sequence of the 54 N-terminal amino acid residues has been determined by automated Edman degradation. This sequence is identical to that of the predicted amino acid sequence of the 20-kd C-terminal part of the colicin A polypeptide deduced from the nucleotide sequence of the caa gene. This polypeptide can produce channels in phospholipid planar bilayers of the same size as those formed by colicin A. However, the voltage-dependence for opening and closing was drastically altered in the peptide fragment channels. The latter, in contrast to colicin A channels, remained open over a wide range of voltage. Large negative potentials were required to close the peptide fragment channels although opening took place in the same voltage range as for colicin A ionic pores.     

Molecular-Level Characterization of Lipid Membrane Electroporation using Linearly Rising Current

We present experimental and theoretical results of electroporation of small patches of planar lipid bilayers by means of linearly rising current. The experiments were conducted on ~120-μm-diameter patches of planar phospholipid bilayers. The steadily increasing voltage across the bilayer imposed by linearly increasing current led to electroporation of the membrane for voltages above a few hundred millivolts. This method shows new molecular mechanisms of electroporation. We recorded small voltage drops preceding the breakdown of the bilayer due to irreversible electroporation. These voltage drops were often followed by a voltage re-rise within a fraction of a second. Modeling the observed phenomenon by equivalent electric circuits showed that these events relate to opening and closing of conducting pores through the bilayer. Molecular dynamics simulations performed under similar conditions indicate that each event is likely to correspond to the opening and closing of a single pore of about 5 nm in diameter, the conductance of which ranges in the 100-nS scale. This combined experimental and theoretical investigation provides a better quantitative characterization of the size, conductance and lifetime of pores created during lipid bilayer electroporation. Such a molecular insight should enable better control and tuning of electroporation parameters for a wide range of biomedical and biotechnological applications.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Patch clamp studies of single intact secretory granules.

The membrane of secretory granules is involved in the molecular events that cause exocytotic fusion. Several of the proteins that have been purified from the membrane of secretory granules form ion channels when they are reconstituted in lipid bilayers and, therefore, have been thought to form part of the molecular structure of the exocytotic fusion pore. We have used the patch clamp technique to study ion conductances in single isolated secretory granules from beige mouse mast cells. We found that the membrane of the intact granule had a conductance of < 50 pS. No abrupt changes in current corresponding to the opening and closing of ion channels were observed, even under conditions where exocytotic fusion occurred. However, mechanical tension or a large voltage pulse caused the breakdown of the granule membrane resulting in the abrupt opening of a pore with an ion conductance of about 1 nS that fluctuated rapidly and could expand to an immeasurably large conductance or close completely. Surprisingly, the behavior of these pores resembled the pattern of conductance changes of exocytotic fusion pores observed in degranulating beige mast cells. This similarity supports the view that the earliest fusion pore is formed upon the breakdown of a bilayer such as that formed during hemifusion.     

Hidden Markov model analysis of intermediate gating steps associated with the pore gate of shaker potassium channels.

Cooperativity among the four subunits helps give rise to the remarkable voltage sensitivity of Shaker potassium channels, whose open probability changes tenfold for a 5-mV change in membrane potential. The cooperativity in these channels is thought to arise from a concerted structural transition as the final step in opening the channel. Recordings of single-channel ionic currents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductance levels in addition to the fully open and closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. Here, we report a similar fine structure in the closing transitions of Shaker channels lacking the mutation. Describing the deactivation time course with hidden Markov models, we find that two subconductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 micros at -100 mV, and the voltage dependences of transitions among these states suggest that they arise from equivalent conformational changes occurring in individual subunits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformational changes in the subunits give rise to changes in a "pore gate" associated with the selectivity filter region of the channel, producing the subconductance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in comparison.     

A cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

In order to determine the mechanism of action of the 27 kDa mosquitocidal delta-endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation-selective channels in the bilayers, permeable to K+ and Na+ but not to N-methylglucamine or Cl-, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid-osmotic lysis.     

Ion channel activity of brain abundant protein BASP1 in planar lipid bilayers

BASP1 (also known as CAP-23 and NAP-22) is a brain abundant myristoylated protein localized at the inner surface of the presynaptic plasma membrane. Emerging evidence suggests that BASP1 is critically involved in various cellular processes, in particular, in the accumulation of phosphatidylinositol-4,5-diphosphate (PIP(2)) in lipid raft microdomains. We have recently shown that BASP1 forms heterogeneously-sized oligomers and higher aggregates with an outward similarity to oligomers and protofibrils of amyloid proteins. However, BASP1 is not known to be related to any amyloid disease. In the present study, we show that BASP1 induces single channel currents across negatively-charged planar lipid bilayers (containing phosphatidylserine or PIP(2)) bathed in 0.1-0.2 M KCl (pH 7.5). By their characteristics, BASP1 channels are similar to amyloid protein channels. BASP1 channels exhibit multiple conductance levels, in the range 10-3000 pS, with the most frequently observed conductance state of approximately 50 pS. The channels demonstrate a linear current-voltage relationship and voltage-independent kinetics of opening and closing. Their K(+) to Cl(-) permeability ratio is approximately 14, indicating that BASP1 channels are cation-selective. The ion channel activity of BASP1 is in accordance with the pore-like structure of BASP1 oligomers observed by electron microscopy on a lipid monolayer. Neuronal protein GAP-43, which is functionally related to BASP1 and also forms oligomers, elicited no ion channel currents under the conditions used in the present study. Elucidation of the physiological or pathological roles of ion channel activity of membrane-bound BASP1 oligomers will help to define the precise mechanism of amyloid protein toxicity.     

Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Discrete conductance fluctuations in lipid bilayer protein membranes

Discrete fluctuations in conductance of lipid bilayer membranes may be observed during the initial stages of membrane interaction with EIM ("excitability inducing material"), during destruction of the EIM conductance by proteolysis, and during the potential-dependent transitions between low and high conductance states in the "excitable" membranes. The discrete conductance steps observed during the initial reaction of EIM with the lipid membranes are remarkably uniform, even in membranes of widely varying lipid composition. They range only from 2 to 6 x 10_-10 ohm_-1 and average 4 x 10_-10 ohm_-1. Steps found during destruction of the EIM conductance by proteolysis are somewhat smaller. The transition between high conductance and low conductance states may involve steps as small as 0.5 x 10_-10 ohm_-1. These phenomena are consistent with the formation of a stable protein bridge across the lipid membrane to provide a polar channel for the transport of cations. T6he uniform conductance fluctuations observed during the formation of these macromolecular channels may indicate that the ions in a conductive channel, in its open state, are largely protected from the influence of the polar groups of the membrane lipids. Potential-dependent changes in conductance may be due to configurational or positional changes in the protein channel. Differences in lipid-lipid and lipid-macromolecule interactions may account for the variations in switching kinetics in various membrane systems.     

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.