Block of neuronal chloride channels by tetraethylammonium ion derivatives

The block by the symmetric tetraethylammonium (TEA) ion derivatives tetrapropylammonium (TPrA), tetrabutylammonium (TBA), and tetrapentylammonium (TPeA) ions of fast chloride channels in acutely dissociated rat cortical neurons was studied with the excised inside- out configuration of the patch-clamp technique. When applied to the intracellular membrane surface, all three of the quaternary ammonium compounds (QAs) induced the appearance of short-lived closed states in a manner consistent with a blocking mechanism where the blocker preferentially binds to the open kinetic state and completely blocks ion current through the channel. The drug must leave the channel before the channel can return to a closed state. The mechanism of block was studied using one-dimensional dwell-time analysis. Kinetic models were fit to distributions of open and closed interval durations using the Q- matrix approach. The blocking rate constants for all three of the QAs were similar with values of approximately 12-20 x 10(6) M-1s-1. The unblocking rates were dependent on the size or hydrophobicity of the QA with the smallest derivative, TPrA, inducing a blocked state with a mean lifetime of approximately 90 microseconds, while the most hydrophobic derivative, TPeA, induced a blocked state with a mean lifetime of approximately 1 ms. Thus, it appears as though quaternary ammonium ion block of these chloride channels is nearly identical to the block of many potassium channels by these compounds. This suggests that there must be structural similarities in the conduction pathway between anion and cation permeable channels.     

Voltage-dependent block of fast chloride channels from rat cortical neurons by external tetraethylammonium ion

Tetraethylammonium ion (TEA) and its longer chain derivatives have been used extensively to block currents through K-selective ion channels. Substantial information has been gained about the structure and gating mechanisms of K and other cation channels from the analysis of the blocking interactions of TEA and other quaternary ammonium ions. We now present an analysis of blocking interactions between single Cl-selective ion channels from acutely dissociated rat cortical neurons and externally applied TEA. TEA applied to the extracellular membrane surface (TEAo) blocked Cl channels in a voltage-dependent manner, with hyperpolarizing potentials favoring block. The voltage dependence of block could be adequately fit assuming that TEA enters the channel pore and binds to a site located approximately 28% of the way through the membrane electrical field. The dose-response relationship between fractional current and [TEA]o at a fixed holding potential of -40 mV was well fit to a simple model with two blocking sites with dissociation constants (Kd) of approximately 2 and 70 mM. The dose-response relationship could also be fit by a mechanism where TEA only partially blocks the channels. At the bandwidth used in these experiments (1-2 kHz), both the mean open duration (composed of the open and blocked durations) and burst duration (composed of open, blocked, and short lifetime shut durations) increased with increased [TEA]o. This is expected if TEAo can bind and unbind only when the channel is in the open kinetic state. These results suggest that the structure of the permeability pathway of these anion-selective channels may be very similar to that of other channels that are blocked by TEA. Additionally, these results caution that a blocking effect by TEA cannot, by itself, be used as sufficient evidence for implicating the participation of K channels in a particular process.     

Subconductance block of single mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics

The activity of single mechanosensitive channels was recorded from cell- attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were designed to investigate the mechanism of channel block produced by externally applied aminoglycoside antibiotics. Neomycin and other aminoglycosides reduced the amplitude of the single-channel current at negative membrane potentials. The block was concentration-dependent, with a half-maximal concentration of approximately 200 microM. At high drug concentrations, however, block was incomplete with roughly one third of the current remaining unblocked. Neomycin also caused the channel to fluctuate between the open state and a subconductance level that was also roughly one third the amplitude of the fully open level. An analysis of the kinetics of the subconductance fluctuations was consistent with a bimolecular reaction between an aminoglycoside molecule and the open channel (kon = approximately 1 x 10(6) M-1s-1 and koff = approximately 400 s-1 at -60 mV). Increasing the external pH reduced both the rapid block of the open channel and the frequency of the subconductance fluctuations, as if both blocking actions were produced by a single active drug species with a pKa = approximately 7.5. The results are interpreted in terms of a mechanism in which an aminoglycoside molecule partially occludes ion flow through the channel pore.     

Kinetics of 9-aminoacridine block of single Na channels

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.     

Coupling of voltage-dependent gating and Ba++ block in the high- conductance, Ca++-activated K+ channel

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.     

Cadmium block of calcium current in frog sympathetic neurons.

Cd2+ blocks whole-cell calcium currents in frog sympathetic neurons by 50% at approximately 300 nM. Strong depolarizations rapidly reverse that blockade (tau = 1.3 ms at +120 mV). Reblocking follows bimolecular kinetics (rate = 1.2 x 10(8) M-1 s-1) at voltages where channels are mostly open (0 to +30 mV). The unblocking rate is approximately 50 s-1, so the dissociation constant calculated from the rate constants is approximately 400 nM. Steady-state block is strong at -80 mV, so closed channels can also be blocked. However, reblocking is extremely slow (tau = 1-2 s) at voltages where the channels are mostly closed. The rates for Cd2+ entry and exit are greater than 100-fold lower for closed channels than for open channels, and closed channels appear to be closed at both ends.     

Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.     

Conformational changes in the pore of CLC-0

The Torpedo Cl- channel, CLC-0, is inhibited by clofibric acid derivatives from the intracellular side. We used the slow gate-deficient mutant CLC-0C212S to investigate the mechanism of block by the clofibric acid-derivative p-chlorophenoxy-acetic acid (CPA). CPA blocks open channels with low affinity (KDO= 45 mM at 0 mV) and shows fast dissociation (koff = 490 s-1 at -140 mV). In contrast, the blocker binds to closed channels with higher affinity and with much slower kinetics. This state-dependent block coupled with the voltage dependence of the gating transitions results in a highly voltage-dependent inhibition of macroscopic currents (KD approximately 1 mM at -140 mV; KD approximately 65 mM at 60 mV). The large difference in CPA affinity of the open and closed state suggests that channel opening involves more than just a local conformational rearrangement. On the other hand, in a recent work (Dutzler, R., E.B. Campbell, and R. MacKinnon. 2003. Science. 300:108-112) it was proposed that the conformational change underlying channel opening is limited to a movement of a single side chain. A prediction of this latter model is that mutations that influence CPA binding to the channel should affect the affinities for an open and closed channel in a similar manner since the general structure of the pore remains largely unchanged. To test this hypothesis we introduced point mutations in four residues (S123, T471, Y512, and K519) that lie close to the intracellular pore mouth or to the putative selectivity filter. Mutation T471S alters CPA binding exclusively to closed channels. Pronounced effects on the open channel block are observed in three other mutants, S123T, Y512A, and K519Q. Together, these results collectively suggest that the structure of the CPA binding site is different in the open and closed state. Finally, replacement of Tyr 512, a residue directly coordinating the central Cl- ion in the crystal structure, with Phe or Ala has very little effect on single channel conductance and selectivity. These observations suggest that channel opening in CLC-0 consists in more than a movement of a side chain and that other parts of the channel and of the selectivity filter are probably involved.     

Nimodipine block of calcium channels in rat vascular smooth muscle cell lines. Exceptionally high-affinity binding in A7r5 and A10 cells

Calcium channel currents were studied in the A10 and A7r5 cell lines derived from rat thoracic aorta muscle cells. The whole-cell variation of the patch voltage clamp technique was used. Results with each cell line were nearly identical. Two types of Ca channels were found in each cell line that are similar to the L-type and T-type Ca channels found in excitable cells. Nimodipine block of the L-type Ca channels in both cell lines is more potent than in previously studied tissues. The kinetics of nimodipine block are accounted for by a model that postulates 1:1 drug binding to open Ca channels with an apparent dissociation constant (KO) of 16-45 pM. In A7r5 cells, the rate of onset of nimodipine block increases with the test potential, in quantitative agreement with the model of open channel block. The apparent association rate (f) is 1.4 x 10(9) M-1 s-1; the dissociation rate (b) is about 0.024 s-1. In anterior pituitary cells (GH4C1 cells), KO is 30 times larger; b is only twice as fast, but f is 15 times slower. The comparative kinetic analysis indicates that the high-affinity binding site for nimodipine is similar in both GH4C1 and A7r5 cells, but nimodipine diffuses much faster or has a larger partition coefficient into the plasmalemma of A7r5 cells than for GH4C1 cells. Unusually high-affinity binding was not observed in earlier 45Ca flux studies with A10 and A7r5 cells. The model of open channel block accounts for the discrepancy; only a small fraction of the Ca channels are in the high affinity open state under the conditions used in 45Ca flux studies, so an effective binding constant is measured that is much greater than the dissociation constant for high-affinity binding.     

The modes of action of gallamine

The action of gallamine, a classical competitive neuromuscular blocking agent, has been examined on voltage-clamped endplates of frog skeletal muscle fibres. Gallamine produces a parallel shift of the equilibrium log (concentration)--response curves in concentrations of up to about 40 microM. At a membrane potential of -70 mV the Schild plot of the dose ratios so measured has a gradient of slightly less than the theoretical value, for a competitive antagonist, of unity. The apparent equilibrium constant for 'competitive' block is about 2 microM, and is approximately independent of the membrane potential. Fluctuation analysis of the endplate current shows two components in the presence of gallamine. The results can be fitted, over the range tested, by a mechanism that involves block of open ion channels by gallamine in a manner similar to that by procaine or quaternary local anaesthetic analogues. The rate constants for this action are strongly dependent on the membrane potential. At -100 mV the association rate constant is about 4 x 10(7) M-1S-1, the dissociation rate constant is about 600 s-1, and the equilibrium constant about 15 microM. Other kinetic measurements (voltage-jump relaxation, and nerve-evoked endplate currents) give results consistent with this conclusion, but apparently these results are valid over a range of conditions narrower than that for fluctuation analysis.     

Dynamic control of deactivation gating by a soluble amino-terminal domain in HERG K(+) channels

K(+) channels encoded by the human ether-à-go-go-related gene (HERG) are distinguished from most other voltage-gated K(+) channels by an unusually slow deactivation process that enables cardiac I(Kr), the corresponding current in ventricular cells, to contribute to the repolarization of the action potential. When the first 16 amino acids are deleted from the amino terminus of HERG, the deactivation rate is much faster (Wang, J., M.C. Trudeau, A.M. Zappia, and G.A. Robertson. 1998. J. Gen. Physiol. 112:637-647). In this study, we determined whether the first 16 amino acids comprise a functional domain capable of slowing deactivation. We also tested whether this "deactivation subdomain" slows deactivation directly by affecting channel open times or indirectly by a blocking mechanism. Using inside-out macropatches excised from Xenopus oocytes, we found that a peptide corresponding to the first 16 amino acids of HERG is sufficient to reconstitute slow deactivation to channels lacking the amino terminus. The peptide acts as a soluble domain in a rapid and readily reversible manner, reflecting a more dynamic regulation of deactivation than the slow modification observed in a previous study with a larger amino-terminal peptide fragment (Morais Cabral, J.H., A. Lee, S.L. Cohen, B.T. Chait, M. Li, and R. Mackinnon. 1998. Cell. 95:649-655). The slowing of deactivation by the peptide occurs in a dose-dependent manner, with a Hill coefficient that implies the cooperative action of at least three peptides per channel. Unlike internal TEA, which slows deactivation indirectly by blocking the channels, the peptide does not reduce current amplitude. Nor does the amino terminus interfere with the blocking effect of TEA, indicating that the amino terminus binding site is spatially distinct from the TEA binding site. Analysis of the single channel activity in cell-attached patches shows that the amino terminus significantly increases channel mean open time with no alteration of the mean closed time or the addition of nonconducting states expected from a pore block mechanism.We propose that the four amino-terminal deactivation subdomains of the tetrameric channel interact with binding sites uncovered by channel opening to specifically stabilize the open state and thus slow channel closing.     

Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.     

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.     

Open channel block by gadolinium ion of the stretch-inactivated ion channel in mdx myotubes.

Currents flowing through single stretch-inactivated ion channels were recorded from cell-attached patches on myotubes from mdx mice. Adding micromolar concentrations of gadolinium to patch electrodes containing normal saline produced rapid transitions in the single-channel current between the fully open and closed states. The kinetics of the current fluctuations followed the predictions of a simple model of open channel block in which the transitions in the current arise from the entry and exit of Gd from the channel pore: histograms of the open and closed times were well fit with single exponentials, the blocking rate depended linearly on the concentration of gadolinium in the patch electrode, and the unblocking rate was independent of the concentration of gadolinium. Hyperpolarizing the patch increased the rate of unblocking (approximately e-fold per 85 mV), suggesting the charged blocking particle can exit the channel into the cell under the influence of the applied membrane field. The rate of blocking was rapid and was independent of the patch potential, consistent with the rate of ion entry into the pore being determined by its rate of diffusion in solution. When channel open probability was reduced by applying suction to the electrode, the blocking kinetics were independent of the extent of inactivation, suggesting that mechanosensitive gating does not modify the structure of the channel pore.     

Blockade of GABAA receptor channels by niflumic acid prevents agonist dissociation

The modulation by the nonsteroidal anti-inflammatory drug niflumic acid (NFA) of the GABAA receptor-mediated currents was studied in acutely isolated cerebellar Purkinje cells using the whole-cell recording and fast drug application system. At concentrations of 3–300 μM NFA potentiated GABA (2 μM)-activated currents, and at concentrations of 1–3 mM NFA blocked these responses. The NFA-induced block was strongly voltage-dependent. Analysis of the voltage dependence of the block suggests that the blocking action of NFA is a result of NFA binding at the site located within GABAA channel pore. The termination of GABA and NFA application was followed by a transient increase of the inward current — “tail” current. These data suggest that NFA acts as a sequential open channel blocker, which prevents dissociation of agonist while the channel is blocked. The tail current develops because, prior to dissociation of agonist, the channels that are in the blocked state must return to the close state via the open state. The tail currents were compared in the presence and absence of gabazine, a competitive antagonist that also allosterically inhibits GABA_A receptors. Application of gabazine only during development of tail current did not change neither amplitude nor time course of this current, while noncompetitive antagonists picrotoxin and penicillin blocked it. Protection of tail current from gabazine block indicates that GABA cannot dissociate from the open-blocked state and the agonist was trapped on the receptor while the channel was open. Trapping was specific for the agonist, because the positive allosteric modulator zolpidem (benzodiazepine site agonist) was able to potentiate the tail current in the absence of GABA in the external solution. Our observations provide a model-independent functional support of the hypothesis that open channel block of GABAA channels by NFA prevents an escape of the agonist from its binding sites.     

Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site

The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.     

Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel.     

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.     

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers.

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.