Interaction of spin-labeled local anesthetics with the sodium channel of squid axon membranes

Summary The effects of spin-labeled local anesthetics on sodium currents of internally perfused squid axons were studied using the voltage-clamp technique. Internal application (10 m) of the most potent spin-labeled local anesthetic used in this study produced a small initial block of sodium currents. However, after sixty repetitive pulses (to +80 mV) given at 1 Hz, the sodium currents were drastically reduced. In addition to this frequency-dependent phenomenon, the anesthetic effect on the sodium currents was also sensitive to the voltage of the pulses. Both the frequency- and voltage-dependent properties remained intact after removal of sodium inactivation with pronase. The recovery of sodium currents from this frequency-dependent anesthetic effect followed a single exponential curve with a surprisingly long time constant of about 10 min. Such a long recovery time, which is longer than any known sodium inactivation process, led us to suggest that the recovery process represents the dissociation of drug molecules from their binding sites. We have also found that increasing hydrophobic character of the homologues series of spin-labeled local anesthetics enhances the frequency- and voltage-dependent block of sodium currents. This effect strongly suggests that hydrophobic interaction is an integral component of the binding site. These probes with their selective effects on the sodium currents, are expected to be highly useful in studying the molecular structure of the sodium channels.     

Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons

The inhibition of sodium currents by local anesthetics and other blocking compounds was studied in perfused, voltage-clamped segments of squid giant axon. When applied internally, each of the eight compounds studied results in accumulating "use-depnedent" block of sodium currents upon repetitive pulsing. Recovery from block occurs over a time scale of many seconds. In axons treated with pronase to completely eliminate sodium inactivation, six of the compounds induce a time- and voltage-dependent decline of sodium currents after activation during a maintained depolarization. Four of the time-dependent blocking compounds--procaine, 9-aminoacridine, N-methylstrychnine, and QX572--also induce altered sodium tail currents by hindering closure of the activation gating mechanism. Treatment of the axon with pronase abolishes use-dependent block completely by QX222, QX314, 9-aminoacridine, and N-methylstrychnine, but only partially be tetracaine and etidocaine. Two pulse experiments reveal that recovery from block by 9-aminoacridine or N-methyl-strychnine is greatly accelerated after pronase treatment. Pronase treatment abolishes both use-dependent and voltage-dependent block by QX222 and QX314. These results provide support for a direct role of the inactivation gating mechanism in producing the long-lasting use-dependent inhibition brought about by local anesthetic compounds.     

Kinetic analysis of phasic inhibition of neuronal sodium currents by lidocaine and bupivacaine.

Phasic ("use-dependent") inhibition of sodium currents by the tertiary amine local anesthetics, lidocaine and bupivacaine, was observed in voltage-clamped node of Ranvier of the toad, Bufo marinus. Local anesthetics were assumed to inhibit sodium channels through occupation of a binding site with 1:1 stoichiometry. A three-parameter empirical model for state-dependent anesthetic binding to the Na channel is presented: this model includes two discrete parameters that represent the time integrals of binding and unbinding reactions during a depolarizing pulse, and one continuous parameter that represents the rate of unbinding of drug between pulses. The change in magnitude of peak sodium current during a train of depolarizing pulses to 0 mV was used as an assay of the extent of anesthetic binding at discrete intervals; estimates of model parameters were made by applying a nonlinear least-squares algorithm to the inhibition of currents obtained at two or more depolarizing pulse rates. Increasing the concentration of drug increased the rate of binding but had little or no effect on unbinding, as expected for a simple bimolecular reaction. The dependence of the model parameters on pulse duration was assessed for both drugs: as the duration of depolarizing pulses was increased, the fraction of channels binding drug during each pulse became significantly larger, whereas the fraction of occupied channels unbinding drug remained relatively constant. The rate of recovery from block between pulses was unaffected by pulse duration or magnitude. The separate contributions of open (O) and inactivated (I) channel binding of drug to the net increase in block per pulse were assessed at 0 mV: for lidocaine, the forward binding rate ko was 1.3 x 10(5) M-1 s-1, kl was 2.4 x 10(4) M-1 s-1; for bupivacaine, ko was 2.5 x 10(5) M-1 s-1, kl was 4.4 x 10(4) M-1 s-1. These binding rates were similar to those derived from time-dependent block of maintained Na currents in nodes where inactivation was incomplete due to treatment with chloramine-T. The dependence of model parameters on the potential between pulses (holding potential) was examined. All three parameters were found to be nearly independent of holding potential from -70 to -100 mV. These results are discussed with respect to established models of dynamic local anesthetic-Na channel interactions.     

Sodium inactivation mechanism modulates QX-314 block of sodium channels in squid axons.

Blocking action of Na channels by QX-314, a quaternary derivative of lidocaine, was studied in internally perfused and voltage-clamped axons of squid. In axons with intact Na inactivation, QX-314 exhibited both a frequency- and a voltage-dependent block of Na channels. Repetitive pulsing to more positive potentials enhanced the degree of block. Both frequency- and voltage-dependent blocks disappeared in axons in which Na inactivation had been destroyed by either pronase or N-bromoacetamide treatment. These results support the notion that Na inactivation not only modulates the frequency-dependent block but also involves the voltage-dependent binding reaction between QX-314 and Na channels.     

Inhibition of sodium currents by local anesthetics in chloramine-T- treated squid axons. The role of channel activation

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.     

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.     

Inhibition of sodium currents in frog ranvier node treated with local anesthetics Role of slow sodium inactivation

Effects of different local anesthetics of sodium permeability were studied in single nerve fibres of frog by the method of voltage clamp. Inhibition of sodium current by externally applied tertiary anesthetics, procaine and trimecaine, was the sum of a potentially independent block (reduced $\text{P}{}_{\mathrm{rmNa}}$) and slow sodium inactivation with time constants ranging from tens to hundreds of ms depending on membrane potential (at room temperature). Externally applied uncharged benzocaine produced a potentially independent block only. According to dose-response curves both processes are one-to-one reactions. In the case of trimecaine equilibrium constant the reaction responsible for reduction of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ is about 0.3 mM, while that for slow inactivation is more than ten times less (0.02 mM). Increasing pH from 5.6 to 8.5 markedly accelerated the slow inactivation process at all potential values. Divalent cations Ca²⁺ and Ni²⁺ shifted the steady-state slow inactivation curve along the potential axis and simultaneously reduced slow inactivation at the saturation level. Permanently charged quaternary trimecaine was ineffective when applied externally. Internally applied tertiary anesthetics and quaternary trimecaine as well as externally applied quaternary derivative of lidocaine QX-572 produced a progressively irreversible block enhanced by depolarization and inhibition reversibly increased by repetitive short-term depolarization (frequency-dependent inhibition). Inhibition of sodium currents by repetitive stimulation observed also in the case of externally applied tertiary anesthetics is due mainly to slow inactivation. The data suggests the existence of several types of receptor sites through which local anesthetics exert their blocking action on sodium permeability.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

Molecular and functional determinants of local anesthetic inhibition of NaChBac

In our recent publication, we describe the local anesthetic (LA) inhibition of the prokaryotic voltage gated sodium channel NaChBac. Despite the numerous functional and putative structural differences with the mammalian sodium channels, the data show that LA compounds effectively and reversibly inhibit NaChBac channels in a concentration range similar to resting blockade on eukaryotic Navs. In addition to current reduction, LA application accelerated channel inactivation kinetics of NaChBac which could be accounted for in a simple state-model whereby local anesthetics increase the probability of entering the inactivated state. We have further explored what state (or states) local anesthetic blockade of NaChBac could pertain to eukaryotic sodium channels, and what molecular similarities exist between these disparate channel families. Here we show that the rate of recovery from inactivation remains unaffected in the presence of local anesthetics. Further, we show that two sites that support use-dependent inhibition in eukaryotic channels, do not affect block to the same extent when mutated in NaChBac channels. The data indicate that the molecular determinants and the inherent mechanisms for LA block are likely to be divergent between bacterial and eukaryotic Navs, but future experiments will help define possible similarities.     

Molecular and functional determinants of local anesthetic inhibition of NaChBac

In our recent publication, we describe the local anesthetic (LA) inhibition of the prokaryotic voltage gated sodium channel NaChBac. Despite the numerous functional and putative structural differences with the mammalian sodium channels, the data show that LA compounds effectively and reversibly inhibit NaChBac channels in a concentration range similar to resting blockade on eukaryotic Navs. In addition to current reduction, LA application accelerated channel inactivation kinetics of NaChBac which could be accounted for in a simple state-model whereby local anesthetics increase the probability of entering the inactivated state. We have further explored what state (or states) local anesthetic blockade of NaChBac could pertain to eukaryotic sodium channels, and what molecular similarities exist between these disparate channel families. Here we show that the rate of recovery from inactivation remains unaffected in the presence of local anesthetics. Further, we show that two sites that support use-dependent inhibition in eukaryotic channels, do not affect block to the same extent when mutated in NaChBac channels. The data indicate that the molecular determinants and the inherent mechanisms for LA block are likely to be divergent between bacterial and eukaryotic Navs, but future experiments will help define possible similarities.     

Retarded recovery of excitability and twitches of frog toe muscles after K-induced depolarization (slow inactivation) in the presence of alkyl amphipaths

Alkyl amphipaths resemble conventional local anesthetics in their ability to retard the recovery of excitability and twitch tension after depolarization at high Ko, an effect that is attributed to slow inactivation of potential-dependent sodium channels. The similar effect of low temperature offers an explanation for its ability to enhance the frequency-dependent effects of these agents.     

Interactions of monovalent cations with sodium channels in squid axon. II. Modification of pharmacological inactivation gating

The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.     

Bupivacaine blocks N-type inactivating Kv channels in the open state: no allosteric effect on inactivation kinetics

Local anesthetics bind to ion channels in a state-dependent manner. For noninactivating voltage-gated K channels the binding mainly occurs in the open state, while for voltage-gated inactivating Na channels it is assumed to occur mainly in inactivated states, leading to an allosterically caused increase in the inactivation probability, reflected in a negative shift of the steady-state inactivation curve, prolonged recovery from inactivation, and a frequency-dependent block. How local anesthetics bind to N-type inactivating K channels is less explored. In this study, we have compared bupivacaine effects on inactivating (Shaker and K_v3.4) and noninactivating (Shaker-IR and K_v3.2) channels, expressed in Xenopus oocytes. Bupivacaine was found to block these channels time-dependently without shifting the steady-state inactivation curve markedly, without a prolonged recovery from inactivation, and without a frequency-dependent block. An analysis, including computational testing of kinetic models, suggests binding to the channel mainly in the open state, with affinities close to those estimated for corresponding noninactivating channels (300 and 280 μM for Shaker and Shaker-IR, and 60 and 90 μM for K_v3.4 and K_v3.2). The similar magnitudes of K_d, as well as of blocking and unblocking rate constants for inactivating and noninactivating Shaker channels, most likely exclude allosteric interactions between the inactivation mechanism and the binding site. The relevance of these results for understanding the action of local anesthetics on Na channels is discussed.     

Fast frequency-dependent block of action potential upstroke in rabbit atrium by small local anesthetics.

Local anesthetics depress the rapid depolarizing phase of atrial action potentials in a frequency-dependent manner. Several local anesthetics were tested in this study at doses that only slightly depressed atrial action potential upstrokes at the spontaneous rate near 2 Hz, but that produced marked reductions in atrial fiber upstrokes when the drive rate was increased to 5 Hz. This frequency-dependent blocking action, especially the rate of development of the block during the 5-Hz stimulus train, is compared for several local anesthetics that have a wide range of lipid solubilities. Although the rate of block development does not correlate well with lipid solubility, it does correlate with molecular weight or size of the local anesthetic molecule. Smaller local anesthetics give faster frequency-dependent blocking. The beta-blocker propranolol also induces a frequency-dependent block of action potential upstroke, with the speed of such block development being predictable on the basis of the molecular weight of propranolol. The design of fast frequency-dependent blockers by using the criterion of smaller molecular size represents an important new structure-activity relation that may very well help in the design of better antifibrillatory drugs.     

Aminopyridines block an inactivating potassium current having slow recovery kinetics.

The blocking action of aminopyridines on an inactivating K current (lKi) in GH3 pituitary cells was studied before and after altering the macroscopic decay of the current with N-bromoacetamide (NBA). The first depolarizing pulse delivered either seconds or minutes after beginning 4-aminopyridine (4AP) application, elicited a current with both a more rapid decay and a reduced peak amplitude. The rapid decay (or time-dependent block) was especially prominent in NBA-treated cells. With continued drug application, subsequent test pulses revealed a stable block of peak current, greater in NBA-treated than control cells. Recovery from block was enhanced by hyperpolarizing holding potentials and by the first depolarizing pulse delivered after prolonged recovery intervals. Unlike aminopyridine block of other K currents, there was no convincing evidence for voltage shifts in activation or inactivation, or for voltage and frequency-dependent unblock. Increasing the open probability of the channels did, however, facilitate the block. Although the behavior of currents in 4AP was suggestive of "open channel block," the block was not produced by 4-aminopyridine methiodide, a positively charged aminopyridine. Moreover, because partial block and recovery occurred without opening the channels we suggest that aminopyridines bind to, or near, this K channel, that this binding is enhanced by opening the channel, and that a conformational change is induced which mimics inactivation. Because recovery from block is enhanced by negative potentials, we suggest that aminopyridine molecules may become "trapped" by inactivation awaiting the slow process of reactivation to escape their binding sites.     

Use-dependent inhibition of Na+ currents by benzocaine homologs.

Most local anesthetics (LAs) elicit use-dependent inhibition of Na+ currents when excitable membranes are stimulated repetitively. One exception to this rule is benzocaine, a neutral LA that fails to produce appreciable use-dependent inhibition. In this study, we have examined the use-dependent phenomenon of three benzocaine homologs: ethyl 4-diethylaminobenzoate, ethyl 4-ethoxybenzoate, and ethyl 4-hydroxybenzoate. Ethyl 4-hydroxybenzoate at 1 mM, like benzocaine, elicited little use-dependent inhibition of Na+ currents, whereas ethyl 4-diethylaminobenzoate at 0.15 mM and ethyl 4-ethoxybenzoate at 0.5 mM elicited substantial use-dependent inhibition--up to 55% of peak Na+ currents were inhibited by repetitive depolarizations at 5 Hz. Each of these compounds produced significant tonic block of Na+ currents at rest and shifted the steady-state inactivation curve (h infinity) toward the hyperpolarizing direction. Kinetic analyses showed that the decaying phase of Na+ currents during a depolarizing pulse was significantly accelerated by all drugs, thus suggesting that these drugs also block the activated channel. The recovery time course for the use-dependent inhibition of Na+ currents was relatively slow, with time constants of 6.8 and 4.4 s for ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate, respectively. We conclude that benzocaine and 4-hydroxybenzoate interact with the open and inactivated channels during repetitive pulses, but during the interpulse the complex dissociates too fast to accumulate sufficient use-dependent block of Na+ currents. In contrast, ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate dissociate slowly from their binding site and consequently elicit significant use-dependent block. A common LA binding site suffices to explain the presence and absence of use-dependent block by benzocaine homologs during repetitive pulses.     

Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms

We examined the kinetics of voltage-dependent sodium currents in cerebellar Purkinje neurons using whole-cell recording from dissociated neurons. Unlike sodium currents in other cells, recovery from inactivation in Purkinje neurons is accompanied by a sizeable ionic current. Additionally, the extent and speed of recovery depend markedly on the voltage and duration of the prepulse that produces inactivation. Recovery is faster after brief, large depolarizations (e.g., 5 ms at +30 mV) than after long, smaller depolarizations (e.g., 100 ms at -30 mV). On repolarization to -40 mV following brief, large depolarizations, a resurgent sodium current rises and decays in parallel with partial, nonmonotonic recovery from inactivation. These phenomena can be explained by a model that incorporates two mechanisms of inactivation: a conventional mechanism, from which channels recover without conducting current, and a second mechanism, favored by brief, large depolarizations, from which channels recover by passing transiently through the open state. The second mechanism is consistent with voltage-dependent block of channels by a particle that can enter and exit only when channels are open. The sodium current flowing during recovery from this blocked state may depolarize cells immediately after an action potential, promoting the high-frequency firing typical of Purkinje neurons.     

Ionic mechanism of action of a spin-labeled local anesthetic on squid axon membranes.

The ionic mechanism of action of a spin-labeled local anesthetic (SLA), 2-[N-methyl-N-(2,2,6,6-tetramethylpiperidonooxyl)]-ethyl 4-ethoxylbenzoate, was studied by means of voltage clamp technique with squid giant axons in comparison with the parent compound without spin label moiety, 2-(N,N-dimethyl)ethyl 4-ethoxylbenzoate (GS-01). Like other local anesthetics, they suppressed both sodium and potassium conductance increases. However, three remarkable differences have been noted between SLA and GS-01: (1) SLA is more effective than GS-01 in suppressing the sodium and potassium conductance increases; (2) SLA induces a potassium inactivation, whereas GS-01 is lacking this ability; (3) SLA has no effect on the time to peak sodium current, whereas GS-01 prolongs it. GS-01 resembles procaine with respect to (2) and (3) above. SLA will become a useful probe for the study of the molecular mechanism of local anesthetic aciton and of ionic channel function.     

Dynamics of 9-aminoacridine block of sodium channels in squid axons

The interactions of 9-aminoacridine with ionic channels were studied in internally perfused squid axons. The kinetics of block of Na channels with 9-aminoacridine varies depending on the voltage-clamp pulses and the state of gating machinery of Na channels. In an axon with intact h gate, the block exhibits frequency- and voltage-dependent characteristics. However, in the pronase-perfused axon, the frequency- dependent block disappears, whereas the voltage-dependent block remains unchanged. A time-dependent decrease in Na currents indicative of direct block of Na channel by drug molecule follows a single exponential function with a time constant of 2.0 +/- 0.18 and 1.0 +/- 0.19 ms (at 10 degrees C and 80 m V) for 30 and 100 microM 9- aminoacridine, respectively. A steady-state block can be achieved during a single 8-ms depolarizing pulse when the h gate has been removed. The block in the h-gate intact axon can be achieved only with multiple conditioning pulses. The voltage-dependent block suggests that 9-aminoacridine binds to a site located halfway across the membrane with a dissociation constant of 62 microM at 0 m V. 9-Aminoacridine also blocks K channels, and the block is time- and voltage-dependent.