Cocaine abuse is a serious medical problem in the USA and

the incidence of cocaine-related deaths is on the rise

(National Institutes on Drug Abuse, 1994). Cocaine has

numerous effects on the heart and coronary vasculature

including vasospasms, ischaemia, myocardial infarction,

arrhythmias and ventricular fibrillation (Billman, 1995).

These actions can be attributed to two prominent effects of

cocaine: an increase in sympathetic stimulation to the

heart and coronary vasculature and a direct inhibition of

cardiac ion channels. The mechanisms that underlie the

cardiotoxic effects of cocaine are not well understood

and are probably related to a combination of both the

sympathomimetic and local anaesthetic properties of this

drug. One of the hallmark effects of cocaine in the heart

is a reduction in conduction velocity that can promote

arrhythmias (Schwartz et al. 1989; Crumb et al. 1990; Kabas

et al. 1990). Sodium channels play a key role in the electrical

excitability of the myocardium and are responsible for the

rapid upstroke of the cardiac action potential. Cocaine has

long been known to reduce the cardiac Na+ conductance

by promoting the voltage-dependent inactivation of

‘sodium carrying units’ (Weidman, 1955). Within the

range of concentrations known to cause acute toxicity in

humans (1–70 mM) (Mittleman & Wetli, 1984), cocaine

produces a characteristic voltage- and frequency-dependent

inhibition of cardiac Na+ current (Crumb & Clarkson,

1990).

Many local anaesthetics and antiarrhythmic drugs are

known to inhibit voltage-gated Na+ channels. The inhibition

produced by these drugs is often enhanced by rapid,

repetitive stimulation or prolonged depolarizations,

indicating that binding of the drug is voltage dependent.

To account for this voltage dependence, it has been

postulated that local anaesthetics preferentially bind to the

open or inactivated states while closed channels are generally

believed to bind these drugs with low affinity (Hille, 1977;

Hondeghem & Katzung, 1977). Recent data supports these

predictions for cocaine where binding affinity has been

shown to increase more than 20-fold as the channels shift

between the closed and inactivated conformations, and

cocaine blocks open batrachotoxin-activated Na+ channels

in planar lipid bilayers (Wang, 1988; Wright et al. 1998).

Cocaine binds to a common site on open and inactivatedhuman heart (Nav1.5) sodium channelsM. E. O’Leary and M. Chahine

Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA, USA and Quebec Heart Institute, Laval HospitalResearch Center, Laval University, Sainte-Foy, Quebec, Canada

The inhibition by cocaine of the human heart Na+ channel (Nav1.5) heterologously expressed in

Xenopus oocytes was investigated. Cocaine produced little tonic block of the resting channels but

induced a characteristic, use-dependent inhibition during rapid, repetitive stimulation, suggesting

that the drug preferentially binds to the open or inactivated states of the channel. To investigate

further the state dependence, depolarizing pulses were used to inactivate the channels and promote

cocaine binding. Cocaine produced a slow, concentration-dependent inhibition of inactivated

channels, which had an apparent KD of 3.4 mM. Mutations of the interdomain III–IV linker that

remove fast inactivation selectively abolished this high-affinity component of cocaine inhibition,

which appeared to be linked to the fast inactivation of the channels. A rapid component of cocaine

inhibition persisted in the inactivation-deficient mutant that was enhanced by depolarization and

was sensitive to changes in the concentration of external Na+, properties that are consistent with a

pore-blocking mechanism. Cocaine induced a use-dependent inhibition of the non-inactivating

mutant and delayed the repriming at hyperpolarized voltages, indicating that the drug slowly

dissociated when the channels were closed. Mutation of a conserved aromatic residue (Y1767) of the

D4S6 segment weakened both the inactivation-dependent and the pore-blocking components of the

cocaine inhibition. The data indicate that cocaine binds to a common site located within the internal

vestibule and inhibits cardiac Na+ channels by blocking the pore and by stabilizing the channels in

an inactivated state.

(Received 21 December 2001; accepted after revision 17 March 2002)

Corresponding author M. E. O’Leary: Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, 1020Locust Street JAH 266, Philadelphia, PA 19107, USA. Email: michael.o’leary@mail.tju.edu

Journal of Physiology (2002), 541.3, pp. 701–716 DOI: 10.1113/jphysiol.2001.016139

© The Physiological Society 2002 www.jphysiol.org

The link between local anaesthetic binding and inactivation

has been tested by exposing Na+ channels to enzymes,

chemical modifiers or toxins that disable inactivation,

which in many cases weakens the inhibition produced

by anaesthetics (Yeh, 1978; Cahalan & Shapiro, 1980;

Moczydlowski, 1986; Wang et al. 1987). Although the data

generally support the concept that inactivation contributes

to anaesthetic binding, the interpretation of the results is

often complicated by the non-specific effects of these

treatments which, in addition to removing inactivation,

may also alter other properties such as the gating of the

channel (Armstrong et al. 1973; Postma & Catterall, 1984;

Wang et al. 2000). More recently, studies have used

mutations of the interdomain III–IV linker, the inactivation

gate, that selectively eliminate the rapid component of Na+

channel inactivation (West et al. 1992). This approach

offers several advantages in that the mutation sites are well

defined and the targeted residues do not appear to

contribute directly to local anaesthetic binding. The III–IV

linker mutations weaken or abolish the inhibition produced

by anaesthetics, consistent with an important role for

inactivation in the binding of these drugs (Bennett et al.1995; Pugsley & Goldin, 1999; Grant et al. 2000).

Residues of the D4S6 segment are believed to be exposed

within the internal vestibule of the Na+ channel pore,

where they contribute to a binding site for local

anaesthetics (Ragsdale et al. 1994). Mutations within the

D4S6 segment weaken the voltage- and use-dependent

inhibition produced by many local anaesthetics including

cocaine (Ragsdale et al. 1994; Qu et al. 1995; Wang et al.1998; Weiser et al. 1999; Wright et al. 1998; Li et al. 1999).

The data are consistent with a local anaesthetic binding site

within the D4S6 region of Na+ channels (Ragsdale et al.1996; Wang et al. 1998). More recent data suggest that

residues of the D1S6 and D3S6 also contribute to local

anaesthetic binding (Nau et al. 1999; Wang et al. 2000;

Yarov-Yarovoy et al. 2001).

In this study, the inhibition by cocaine of human cardiac

(hH1, Nav1.5) Na+ channels expressed in Xenopus oocytes

was investigated. Mutations within the D4S6 segment, the

putative local anaesthetic binding site, and interdomain

III–IV linker were used to define further the state-dependent

binding of this drug. Two components of cocaine binding

were detected. A slow component of cocaine inhibition

was observed in the wild-type channel that was linked to

the rapid inactivation of the channels. A rapid component

of cocaine inhibition was observed in the inactivation-

deficient mutant that had properties consistent with a

pore-blocking mechanism. Mutations of the D4S6 segment

weakened both the inactivation-dependent and pore-

blocking components, consistent with a common, high-

affinity binding site for cocaine located within the internal

vestibule of the channel.

METHODS Site-directed mutagenesisAmino acid substitutions were made using the QuickChange Site-Directed Mutagenesis Kit (Stratagene Inc., La Jolla, CA, USA).Fragments (1–2 kb) encompassing the mutation sites were excisedand subcloned into pcDNA. Complementary pairs of oligo-nucleotides (22–29 bp) containing the appropriate nucleotidesubstitutions were prepared at the Nucleic Acid Facility, KimmelCancer Center (Thomas Jefferson University, Philadelphia, PA,USA). These oligonucleotides were subsequently used as primersfor the complete synthesis of both strands of the plasmid. We used20 ng of cDNA plasmid as template, 5 U Pfu DNA polymerase(Stratagene, Inc.), primers, and free nucleotides in a total volumeof 100 ml. After strand synthesis (~20 cycles), 10 U of Dpn I wasadded to the reaction mixture to digest the original methylatedplasmid template (37 °C, 1–2 h). The restriction endonuclease washeat inactivated (65 °C, 15 min), and the mixture used to transformDH5a cells (Gibco BRL) by electroporation. Base substitutionswere confirmed by automated DNA sequencing by the NucleicAcid Facility of the Kimmel Cancer Center at Jefferson MedicalCollege. DNA fragments (1–2 kb) carrying the mutation werethen sub-cloned into wild-type Nav1.5 background and amplifiedin DH5a.

Oocyte expression and two-electrode voltage clampThe cDNA encoding the human cardiac (Nav1.5) Na+ channel inthe pcDNA plasmid (Invitrogen) was linearized with Xba I andfull length capped mRNA transcribed using the T7 promoter(mMessage mMachine, Ambion, Austin, TX, USA). Oocytes wereharvested from mature female Xenopus laevis (Xenopus I, AnnArbor, MI, USA). The animals were anaesthetized by immersionin tricaine (1.5 mg ml_1) and several ovarian lobes surgicallyremoved under sterile conditions. The adhering follicle cell layerwas removed by incubating oocytes with 1 mg ml_1 collagenase(Sigma Chemical, St Louis, MO, USA) in calcium-free OR2(82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 5 mM Hepes, pH 7.4)solution for 2 h. The oocytes were washed with calcium-freeOR2 and transferred to 70 % Leibovitz L-15 medium (LifeTechnologies) supplemented with 15 mM Hepes (pH 7.4), 5 mM

L-glutamine and 10 mg ml_1 gentamycin. Stage IV–V oocyteswere microinjected with 50 nl of cRNA (1–2 mg ml_1) andincubated for 24–48 h at 18 °C. The animals were treated inaccordance with the NIH guidelines and the protocol wasapproved by the Animal Use and Care Committee of ThomasJefferson University.

The currents of cRNA-injected oocytes were recorded using astandard two-electrode voltage clamp technique. Oocytes wereimpaled with microelectrodes (< 1 MV) filled with 3 M KCl andcurrents recorded using an OC-725C voltage clamp (WarnerInstruments, Hamden, CT, USA). Oocytes were held at _100 mVand pulses generated using pCLAMP software (Version 7, AxonInstruments, Foster City, CA, USA). ND96 recording salinecontained (mM): 116 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes,pH 7.4. All recordings were performed at room temperature (23 °C).

Patch clamp recordingsExcised-patch recordings were obtained using an Axopatch 200Apatch clamp amplifier equipped with a DigiData 1200 interface(Axon Instruments). Voltage pulses were generated and datacollected using pCLAMP (Version 7, Axon Instruments). Patchpipettes were fashioned from Corning 8161 glass (Dow Corning)with resistances of 0.5–2 MV and were Sylgard coated to reduce

M. E. O’ Leary and M. Chahine702 J. Physiol. 541.3

capacity transients. Extracellular solution contained (mM): 140choline chloride, 2 KCl, 1.5 CaCl2, 1 MgCl2 and 10 Hepes, pH 7.4with KOH. Pipette solution consisted of (mM): 140 NaCl, 5 EGTAand 10 Hepes, pH 7.4 with NaOH. Patches were held at _100 mVand pulsed to +40 mV for 100 ms at a frequency of 0.2 Hz.Reduced external Na+ solutions (Figs 6, 7 and 9) were prepared byisosmotically replacing NaCl with choline chloride. Cocaine-HClwas purchased from the Jefferson Hospital Pharmacy and preparedas a 50 mM stock solution in water.

RESULTS Cocaine produced a use-dependent inhibition ofNav1.5 Na+ channelsAt low concentrations (< 100 mM) cocaine had little effect

on the current of human cardiac Na+ channels (Nav 1.5)

when the oocytes were held at a hyperpolarized voltage

(_100 mV) and stimulated at low frequency (0.1 Hz).

Under these conditions, cocaine produced little tonic block

of resting channels and any channels that were inhibited

during the depolarizing pulses fully recovered during the

prolonged rest interval. By contrast, the cocaine inhibition

significantly increased during rapid repetitive stimulation,

a phenomenon commonly referred to as use-dependent

inhibition. The use-dependent inhibition was measured

by applying a series of 50 depolarizing pulses to _10 mV at

a frequency of 5 Hz (Fig. 1A). In the absence of the drug,

the channels were capable of efficiently cycling between

the closed, open and inactivated conformations with no

reduction in current amplitude (Fig. 1C). Following the

bath application of 50 mM cocaine, the rapid pulsing protocol

caused a progressive reduction in the amplitude of the

currents, reflecting the cocaine inhibition of the channels.

During the depolarizing pulses, a small fraction the channels

entered into a drug-modified state(s) from which they did

not appreciably recover during the short hyperpolarization

(_100 mV, 180 ms) between pulses. The cocaine inhibition

progressively increased for subsequent depolarizations

within the pulse train as the channels accumulated in this

slowly repriming state.

Residues of the S6 segment of the fourth homologous

domain (D4S6) of brain Na+ channels are known to

contribute to the binding of local anaesthetics (Ragsdale etal. 1994). The homologous D4S6 residues I1756, F1760 and

Y1767 of Nav 1.5 were replaced with cysteines and the effects

of these substitutions on the use-dependent inhibition

were determined. As with the wild-type channel, rapid

repetitive pulsing produced no change in the amplitude of

the currents in the absence of cocaine (Fig. 1D–F). The

use-dependent inhibition of the I1756C mutant measured

in the presence of 50 mM cocaine was only slightly reduced

by comparison to the wild-type channel. By contrast, the

F1760C and Y1767C mutations substantially reduced

or completely abolished the use-dependent inhibition

produced by 50 mM cocaine, suggesting that these residues

may contribute to cocaine binding.

Time course of the cocaine inhibitionAlthough the repetitive pulsing experiments confirmed

that the activation of the channels enhanced the cocaine

inhibition, these protocols caused channels to rapidly cycle

through the closed, open, and inactivated conformations.

Identifying the state(s) important for cocaine binding is

difficult from these types of experiments. To investigate

further the requirements for cocaine binding, we employed

a protocol that effectively stabilized the channels in

inactivated states. Conditioning pulses to _10 mV of

variable duration (1 ms–60 s) were used to inactivate the

channels and promote cocaine binding. A 150 ms pulse to

_100 mV was then applied to allow the recovery from

inactivation and a test pulse was used to assay availability.

The short hyperpolarization that preceded the test pulse

was sufficient to allow for the full recovery of the fast

inactivated (tF = 11 ms) but not cocaine-modified channels

(tI = 1020 ms, tS = 7497 ms; Fig. 4). The test current

amplitudes were normalized to similar currents measured

after a prolonged (60 s) rest at _100 mV and plotted versus

Cocaine inhibition of Nav1.5 Na+ channelsJ. Physiol. 541.3 703

Figure 1. Use-dependent inhibition of Nav1.5 Na+

channels expressed in Xenopus oocytesThe use-dependent inhibition was induced by a series of 50depolarizing pulses to _10 mV for 20 ms applied at a frequency of5 Hz. A and B, currents of the wild-type (A) and F1760C mutant(B) after bath application of 50 mM cocaine. Current tracescorrespond to the pulse numbers 1, 5, 10, and 50 of the stimulationtrain. C–F, the peak current elicited by each pulse within the trainwas normalized to the current of the first pulse and plotted versusthe pulse number. The normalized currents were measured before(filled symbols) and after (open symbols) application of 50 mM

cocaine for wild-type (n = 7), I1756C (n = 4), F1760C (n = 14),and Y1767C (n = 7).

the prepulse duration (Fig. 2A). In the absence of cocaine,

the currents elicited by the test pulses progressively

decreased with conditioning pulses longer than 1 s, and

reached a steady-state level of 55 % of the control current.

The decay of the current was biexponential with time

constants of 158 ms (t1) and 6461 ms (t2), reflecting the

slow inactivation of the channels. Shortening the

hyperpolarizing prepulse that preceded the test pulses

slightly increased the relative amplitude (A1) but did not

alter the time constants of the fast or slow components

(data not shown).

Application of cocaine (5–250 mM) accelerated the time

course of the current decay and decreased the steady-

state current amplitude (Fig. 2A). The majority of the

cocaine inhibition occurred after the channels had

rapidly inactivated (t ∆ 5 ms) but before the onset of

slow inactivation (t = 6.5 s). The time course of the

current decay was fitted by the sum of two exponentials

and the time constants plotted versus the cocaine

concentration (Fig. 2B). At low concentrations (< 50 mM),

cocaine caused a concentration-dependent decrease in the

time constants of the fast (t1) and slow (t2) components of

current decay. At higher concentrations (≥ 100 mM), the

slow component (t2) was no longer detected, the time

constant of the intermediate component (t1) was further

reduced, and a new rapid component of cocaine inhibition

was observed (tf = 6–9 ms). This rapid component of

inhibition has been observed previously in native cardiac

Na+ currents and is believed to reflect the binding of

cocaine to open channels (Crumb & Clarkson, 1990).

When cells were held at a hyperpolarized voltage (_100 mV),

cocaine (< 100 mM) produced little change in the amplitude

of the peak Na+ current elicited by short test pulses,

indicating that closed channels bind cocaine with relatively

low affinity. This contrasts with the inhibition observed

after prolonged depolarization to _10 mV, where 5 mM

cocaine produced a 50 % reduction in the steady-state

current amplitude (Fig. 2A). Depolarization enhanced the

cocaine inhibition consistent with an important role for

inactivation in drug binding. At least two components of

slow inactivation and three components of cocaine

inhibition contributed to the time-dependent decay of the

currents observed in these experiments. Unfortunately,

the extensive overlap of the kinetics of inactivation and the

cocaine inhibition significantly complicates attempts to

correlate directly drug binding with the fast or slow

inactivated states of the channel. Despite the relatively

complex kinetics, an unbiased assessment of the cocaine

inhibition can be obtained by plotting the normalized

reduction in steady-state current amplitude (AW) versusthe concentration (Fig. 2C). Cocaine reduced the steady-

state current with an apparent KD of 3.4 ± 0.4 mM, which is

consistent with previous estimates of cocaine binding to

inactivated Na+ channels (Crumb & Clarkson, 1990; Wright

et al. 1997).

The effect of the D4S6 mutations on the time course of

cocaine inhibition was also investigated. The kinetics of

the current decay and the steady-state amplitude of the

M. E. O’ Leary and M. Chahine704 J. Physiol. 541.3

Figure 2. Time course of cocaine binding to inactivatedchannelsA, the time course of cocaine inhibition was measured using a triplepulse protocol consisting of a conditioning pulse to _10 mV ofvariable duration (1 ms–60 s), a short hyperpolarization to_100 mV for 150 ms and a test pulse to _10 mV. the peak currentselicited by test pulses were normalized to controls (I/Io) and plottedversus the prepulse duration. The decay of the current is bestdescribed by the sum of two exponentials :

I/Io = A1exp(_t/ t1) + A2exp(_t/ tS) + AW,

where A1, A2 and AW are the relative amplitudes of the fast, slow andsteady state components, respectively. The fast (t1) and slow (t2)time constants of 158 ± 76 and 6461 ± 932 ms for control (n = 11),1254 ± 737 and 6284 ± 1559 ms for 5 mM (n = 5), 855 ± 139 and3494 ± 1592 ms for 50 mM (n = 6), 6 ± 4 and 581 ± 21 ms for100 mM (n = 11), 8 ± 6 ms and 267 ± 25 ms (n = 5) for 250 mM

cocaine. B, time constants of the fast, intermediate, and slowcomponents of current decay plotted versus the cocaineconcentration (see text). C, plot of the normalized steady-statecurrent amplitude (AW) versus the cocaine concentration. Thesmooth curve is a fit to a single site model with a KD of 3.4 ± 0.4 mM.

F1760C and Y1767C mutants measured in the presence of

50 mM cocaine were not substantially different from their

respective drug-free controls (Fig. 3B and C). At low

concentrations, cocaine either failed to bind or no longer

inhibited the channels. Increasing the concentration of

cocaine to 250 mM reduced the time constants and the

steady-state current amplitudes of the F1760C and Y1767C

mutants with respect to controls. High concentrations

partially restored the cocaine inhibition, indicating that

the binding was reduced but not abolished by the F1760C

and Y1767C mutations. With 250 mM cocaine, the onset of

the inhibition of the F1760C (t1 = 120 ms, t2 = 3.2 s)

and Y1767C mutants (t1 = 70 ms, t2 = 5.3 s) was slow

by comparison to the wild-type channel (t1 = 8 ms,t2 = 267 ms). The slower onset and the reduced steady-

state inhibition suggest that the F1760C and Y1767C

mutations may disrupt cocaine binding. This contrasts

with the I1756C mutation, which had little effect on the

time course of cocaine inhibition.

Repriming of cocaine-modified channelsThe use-dependent inhibition indicated that a fraction of

the wild-type channels that bind cocaine during the

depolarizing pulses failed to recover during the short

interval between depolarizations. We therefore directly

measured the effect of cocaine on the time course of

repriming using a standard double pulse protocol (Fig. 4).

In the absence of drug, the time course of the recovery is

best fitted with the sum of three exponentials with time

constants of 11, 189 and 1967 ms (Table 1). After application

of 50 mM cocaine, the time constants were 11, 1017 and

7497 ms with the majority of the channels (65 %) recovering

with the slowest time constant. Cocaine delayed the

repriming of the channels, selectively increasing the time

constants of the intermediate and slowest components.

Although the fractional amplitude of the rapid component

was reduced 70 % by cocaine, the time constant was not

altered, suggesting that this rapid component may reflect

the recovery of drug-free channels.

The F1760C and Y1767C mutations either severely

attenuated or completely abolished the cocaine-induced

delay in repriming. In both cases, the recovery time

constants measured in the presence of 50 mM cocaine were

similar to those of the drug-free controls, suggesting that

cocaine either dissociates rapidly at hyperpolarized voltages

or does not appreciably bind during the depolarizing

prepulses. To ensure that the channels were modified by

the drug, the recovery was also measured after applying

250 mM cocaine, a concentration previously shown to

inhibit these mutant channels (Fig. 3). Cocaine reduced

the fraction of F1760C and Y1767C current recovering

with the fast time constant by 73 % and 44 %, respectively,

consistent with an increase in cocaine binding. Despite the

high concentration, the recovery of the drug-modified

F1760C and Y1767C mutant channels was considerably

more rapid than that of the wild-type (Table 1). This rapid

repriming did not appear to result from a non-specific

effect on the stability of the inactivated states, since in the

absence of cocaine the recovery from inactivation of the

mutants was not substantially different from that of the

wild-type. Rather, the data suggest that these mutations

Cocaine inhibition of Nav1.5 Na+ channelsJ. Physiol. 541.3 705

Figure 3. Effects of D4S6 mutations on cocaine inhibitionThe onset of cocaine inhibition was measured using the samepulsing protocol as in Fig. 2. In the absence of cocaine, thedevelopment of slow inactivation was biexponential with fast andslow time constants of 0.6 ± 0.1 and 7.6 ± 0.9 s for I1756C(panel A; A1 = 0.13, n = 7), 1.2 ± 0.5 and 13.8 ± 3.8 s for F1760C(panel B; A1 = 0.22, n = 11), 1.4 ± 0.9 and 10.6 ± 2.1 s for Y1767C(panel C; A1 = 0.10, n = 17). After application of 50 mM cocaine thetime constants were 0.9 ± 0.2 and 7.3 ± 5.2 s for I1756C (A1 = 0.65,n = 4), 1.3 ± 0.4 and 13.3 ± 4.4 s for F1760C (A1 = 0.29, n = 10)and 0.7 ± 0.3 and 7.5 ± 1.8 s Y1767C (A1 = 0.2, n = 7). Thefraction of non-inhibited current (AW) before and after addition of50 mM cocaine was 0.51 ± 0.01 and 0.171 ± 0.02 for I1756C,0.34 ± 0.04 and 0.31 ± 0.04 for F1760C, and 0.54 ± 0.01 and0.43 ± 0.02 for Y1767C. Also plotted is the inhibition of theF1760C and Y1767C mutants by 250 mM cocaine with fast and slowtime constants of 0.12 ± 0.01 and 3.2 ± 0.9 s for F1760C(A1 = 0.65, n = 5) and 0.07 ± 0.02 s and 5.3 ± 0.4 s for Y1767C(A1 = 0.18, n = 4).

cause cocaine to dissociate rapidly from the D4S6 binding

site at hyperpolarized voltages. The slow dissociation of

cocaine from inactivated channels appears to be an

important determinant of the repriming kinetics of drug-

modified channels.

Cocaine inhibition of inactivation-deficientchannelsThe contribution of fast inactivation to the cocaine

inhibition was further investigated using an inactivation-

deficient mutant channel in which a series of hydrophobic

residues of the inter-domain III–IV linker (IFM) were

replaced with glutamines (West et al. 1992). Consistent

with the selective removal of fast inactivation, the currents

of the I1485Q, F1486Q, M1487Q triple mutant of Nav 1.5

(QQQ), rapidly activated but only slowly inactivated

during 2 s depolarizations (Fig. 5). In the absence of drug,

the decay of the current was biexponential with time

constants of 85 and 1063 ms, respectively, reflecting the

slow inactivation of the QQQ mutant channels. Application

of 50 mM cocaine induced a rapid relaxation that is best

fitted by the sum of three exponentials with time constants

of 15, 181 and 1519 ms and the effect of cocaine was

completely reversed upon washout. Cocaine induced a

new, rapid component of current decay and appeared to

slow the onset of the intermediate and slow components.

At concentrations < 100 mM, cocaine only slightly reduced

the peak amplitude of the current, indicating that at low

concentrations few channels are inhibited under resting

conditions. The majority of the cocaine inhibition developed

after the channels had opened, suggestive of a simple pore-

M. E. O’ Leary and M. Chahine706 J. Physiol. 541.3

Table 1. Time course of the recovery from inactivation and cocaine inhibition

t1 (ms) t2 (ms) t3 (ms) A1 A2 A3

Wild-typeControl 11 ± 2 189 ± 150 1967 ± 872 0.58 ± 0.04 0.19 ± 0.06 0.23 ± 0.0750 mM 11 ± 2 1017± 327 7497 ± 560 0.17 ± 0.01 0.19 ± 0.04 0.65 ± 0.04

I1756CControl 12 ± 2 189 ± 103 3074 ± 1168 0.52 ± 0.05 0.25 ± 0.05 0.23 ± 0.0550 mM 23 ± 8 1190 ± 339 7882 ± 1202 0.21 ± 0.04 0.33 ± 0.07 0.47 ± 0.07

F1760CControl 5 ± 1 255 ± 80 2363 ± 1024 0.41 ± 0.03 0.37 ± 0.06 0.22 ± 0.0750 mM 4 ± 1 242 ± 70 2498 ± 361 0.34 ± 0.02 0.25 ± 0.04 0.41 ± 0.04250 mM 10 ± 3 348 ± 123 2569 ± 179 0.11 ± 0.01 0.18 ± 0.04 0.71 ± 0.04

Y1767CControl 4 ± 1 118 ± 99 1395 ± 327 0.46 ± 0.04 0.16 ± 0.06 0.39 ± 0.0650 mM 3 ± 1 76 ± 62 1121 ± 293 0.40 ± 0.07 0.19 ± 0.07 0.41 ± 0.07250 mM 10 ± 3 343 ± 65 4350 ± 1013 0.26 ± 0.03 0.46 ± 0.04 0.27 ± 0.04

The time constants (t) and relatively amplitudes (A) were determined from fits of the recovery data to thesum of three exponentials (Fig. 4). The data are shown before (Control) and after bath application of 50 or250 mM cocaine. The data are the means ± standard errors. Wild-type, n = 7; I1756C, n = 3; F1760C, n = 8;Y1767C, n = 9.

Figure 4. Time course of recoveryfrom cocaine inhibitionChannels were depolarized for 10 s to_10 mV before returning to _100 mV forintervals ranging from 1 ms to 30 s. A_10 mV test pulse was then used to assay thefraction of recovered current, which wasnormalized to control test currents andplotted versus the recovery interval. Therecovery time course was measured before(0) and after bath application of 50 mM (9)(A–D) or 250 mM (2) (C and D)cocaine.The smooth curves are fits to the sum ofthree exponentials with the parameterslisted in Table 1.

blocking mechanism. The onset of the cocaine inhibition

increased in a concentration-dependent fashion with the

rapid component reflecting the channel block (Fig. 5B).

Assuming a simple bimolecular interaction predicts that

the apparent blocking rate (t_1) should increase linearly

with concentration where the slope and y-intercept are

the association (kon) and dissociation (koff) rate constants

(O’Leary et al. 1994). In this experiment, the kon and koff

were 4.7 w 105M

_1 s_1 and 57.9 s_1, respectively, yielding an

estimate of the inhibition constant (KD) at _10 mV of

122 mM (Fig. 5C).

Cocaine block was sensitive to changes in external[Na+]The rapid onset and simple bimolecular kinetics suggest

that cocaine may inhibit the non-inactivating mutant

channels by a pore-blocking mechanism. A common

feature of many pore blockers of Na+ channels is that the

time course and extent of the inhibition is often sensitive

to changes in the concentration of external Na+ (Shapiro,

1977; Cahalan & Almers, 1979). This is believed to result

from competition between the blocker and Na+ ions for

binding sites within the pore. If external Na+ ions and

cocaine compete for a common or overlapping binding

site then a more rapid and extensive block is predicted, as

the concentration of external Na+ is reduced. To test for

trans side effects of Na+ ions, the cocaine blocking kinetics

were compared in external Ringer solution containing

either 100 % (116 mM) or 25 % (29 mM) Na+. With 100 %

external [Na+] cocaine produced a characteristic time-

dependent decay of the current (Fig. 6A). Reducing the

external [Na+] by 75 % increased both the rate of the

current decay and the steady-state inhibition, consistent

with the predictions of a competitive interaction (Fig. 6B).

The blocking rates in normal and low external Na+ Ringer

solution were determined from exponential curve fits

of the current decay and plotted versus the cocaine

concentration (Fig. 6C). Lowering the concentration of

external Na+ caused an increase in the slope (kon) and a

reduction in the y-intercept (koff), consistent with more

rapid binding and slower dissociation. The KD measured at

_25 mV was reduced from 214 mM in 100 % Na+ Ringer

solution to 43 mM in 25 % external Na+ Ringer solution,

indicating that the affinity of the cocaine block is highly

sensitive to changes in the concentration of external Na+.

The data are consistent with a model in which external Na+

ions and cocaine compete for binding sites. The cocaine

binding site appears to be located on the cytoplasmic side

of the channel, suggesting that the competition between

cocaine and external Na+ probably occurs within the

narrow confines of the pore.

Use-dependent inhibition in absence of fastinactivationSimilar to what was observed with the wild-type channel,

rapid repetitive pulsing of the QQQ mutant in the presence

of cocaine produced a use-dependent inhibition (Fig. 7A).

In the absence of drug, the amplitude of the current was

not altered by this rapid pulsing protocol, indicating that

the channels fully recovered during the short hyper-

polarization between pulses. Application of 50 mM cocaine

caused a progressive decrease in the amplitude of the QQQ

mutant current that is typical of use-dependent inhibition.

The onset of this inhibition was more rapid but less

extensive than that observed for wild-type channels

measured under the same conditions (Fig. 7A, dotted

line). The steady-state inhibition measured after 50 pulses

Cocaine inhibition of Nav1.5 Na+ channelsJ. Physiol. 541.3 707

Figure 5. The time course of cocaine block of the QQQmutant channelsThe inactivation-deficient mutant (IFMåQQQ) was constructedby replacing a series of hydrophobic residues of the interdomainIII–IV linker with glutamines (see text). A, currents were elicited bydepolarizing for 2 s to _10 mV from a holding potential of_100 mV. In the absence of cocaine, the decay of the current isbiexponential with time constants of 85.6 ± 6.2 and1063.4 ± 98.4 ms (n = 6). After application of 50 mM cocaine thedecay of the current was fitted with the sum of three exponentialswith time constants of 15.2 ± 0.6, 181.5 ± 7.0 and1519.3 ± 85.7 ms (n = 6). The cocaine inhibition was completelyreversible upon washout with time constants of 75.4 ± 6.8 and1154.6 ± 180.5 ms (n = 6). B, the time course of cocaine inhibitionduring 200 ms depolarizations was measured over a range ofconcentrations and the decay time constants determined frombiexponential curve fits. C, the apparent blocking rate (1/ tf)plotted versus the cocaine concentration is linear with slope (kon)and y-intercept (koff) of 4.7 w 105

M_1 s_1 and 57.9 s_1, respectively.

The inhibition constant (KD = koff/kon) at _10 mV is 122 mM.

was enhanced by lowering the external concentration of

Na+ and is consistent with previous data showing that Na+

ions compete with cocaine for binding sites.

The reduction in the amplitude of the current elicited by

successive depolarizations within the pulse train suggests

that a fraction of the cocaine-blocked channels fail to

recover during the short hyperpolarization between the

pulses. The amplitudes of the currents progressively

decreased as channels accumulated in this drug-modified,

non-conducting state. The QQQ mutant did not inactivate

during short (20 ms) depolarizations, suggesting that

cocaine slowly dissociates when the channels are closed.

To test this mechanism, the time course of the recovery of

cocaine-blocked channels was measured using a double

pulse protocol (Fig. 7B). In the absence of drug, the

recovery had a time constant of 49 ms and fractional

amplitude of 0.91, reflecting the slow inactivation of the

channels. In the presence of 150 mM cocaine the recovery

time course is well fitted by a single exponential with a time

constant of 1.8 s. Channels blocked by cocaine recovered

slowly at _100 mV by comparison with the drug-free

controls. Because cocaine acts by blocking the open

channels, and these channels do not inactivate, our data

suggest that the drug may become trapped within the pore

or the binding is further stabilized as the channels close.

Role of the D4S6 segment in the cocaine block ofnon-inactivating channelsThe D4S6 residues F1760 and Y1767 appear to play a

prominent role in cocaine binding to the inactivated

channel (Figs 3 and 4). In this section, we examine the

effects of these same D4S6 mutations on the cocaine

block of open channels by transferring the I1756C and

Y1767C mutations to the QQQ mutant background. The

F1760C/QQQ mutant was also constructed but failed to

M. E. O’ Leary and M. Chahine708 J. Physiol. 541.3

Figure 6. Cocaine block is sensitive to changes in theconcentration of external Na+

A, whole-cell currents of the QQQ mutant measured in normalRinger solution (116 mM Na+) before and after the application100 mM cocaine. Currents were activated by depolarizing to_25 mV from a holding potential of _100 mV. B, same pulsingprotocol was applied in reduced external Na+ Ringer solution(29 mM Na+). C, the blocking rates were determined from the rapiddecay of the currents as described previously. The association anddissociation rate constants were 3.1 w 105

M_1 s_1 and 66.3 s_1 for

100 % Na+ Ringer solution and 6.3 w 105M

_1 s_1 and 27.3 s_1 for the25 % Na+ conditions. The KD of cocaine inhibition at _25 mV is214 mM in the high external Na+ and 43 mM in low external Na+

Ringer solution.

Figure 7. Use-dependent inhibition of the QQQ mutantNa+ channelsA, a series of 50 depolarizing pulses to _10 mV for 20 ms wereapplied at a frequency of 5 Hz. Currents elicited by individualpulses were normalized and plotted versus pulse number. The use-dependent inhibition was measured in either control 100 % Na+

Ringer solution (116 mM) or 25 % external Na+ Ringer solution(29 mM). The dotted line is the cocaine inhibition of the wild-typechannel in 100 % Na+ solution replotted from Fig. 1C. B, reprimingtime course of cocaine-blocked channels. Cells were depolarizedfor 100 ms to _10 mV in the absence and presence of 150 mM

cocaine before returning to _100 mV for a variable duration(1 ms–30 s). A standard _10 mV test pulse was used to assayavailability after the completion of the recovery interval. In thepresence of cocaine the recovery time course is well fitted by asingle exponential with a time constant of 1.8 ± 0.1 s and a steady-state amplitude of 0.43 ± 0.05 (n = 8). Also plotted is the timecourse of recovery from slow inactivation which has a timeconstant of 49.3 ± 5.0 ms and a steady-state current amplitude of0.91 ± 0.01 (n = 9).

express current. In the absence of cocaine, the slow

inactivation of the QQQ mutant (tf = 58.1 ± 9.5 ms,ts = 913.5 ± 407.6 ms, n = 5) was similar to that of the

I1756C/QQQ (tf = 39.8 ± 1.3 ms, ts = 741.0 ± 54.5 ms,

n = 6) and Y1767C/QQQ mutants (tf = 41.8 ± 4.3 ms,ts = 1091 ± 221.3 ms, n = 6) (Fig. 8).

The blocking kinetics was assessed from the time course of

the cocaine-induced current decay as described previously.

The I1756C mutation slowed both the binding and

unbinding of cocaine in comparison to the controls

(dotted line) and slightly increased the affinity of cocaine

block (KD = 100 mM). By contrast, the Y1767C mutation

completely abolished the cocaine block at concentrations

< 250 mM with the majority of the steady-state reduction at

this higher concentration being attributed to tonic block.

Y1767 appears to play a significant role in cocaine binding

to open channels.

Voltage sensitivity of the cocaine inhibition of theQQQ mutantThe kinetics and voltage sensitivity of the cocaine

inhibition of the QQQ mutant was consistent with an

open-channel blocking mechanism with an apparent KD of

122 mM at _10 mV. This affinity is lower than that observed

for inactivated channels (KD = 3.4 mM) and is slightly

higher than the concentrations of cocaine (0.4–70 mM)

detected in the serum of humans suffering from cocaine

toxicity (Mittleman & Wetli, 1984). The role of the open-

channel block in the cocaine inhibition of Na+ channels invivo is unclear. However, previous studies have shown that

the inhibition produced by many Na+ channel pore

blockers increases with strong depolarization, which is

believed to originate from an electrostatic interaction

between the positively charged drug and the membrane

electric field (Strichartz, 1973). We were therefore interested

in determining the kinetics of cocaine inhibition at +40 mV,

a voltage considered to be near the peak of the cardiac

action potential. Unfortunately, technical difficulties

associated with voltage clamping large oocytes prevented

us from measuring the cocaine block of the QQQ mutant

at positive voltages. However, the cocaine block at more

depolarized voltages can be measured in outside-out

macropatches in which the internal concentration of Na+

has been increased to facilitate the measurement of the

Cocaine inhibition of Nav1.5 Na+ channelsJ. Physiol. 541.3 709

Figure 8. Effects of D4S6 mutations on cocaine blockThe I1756C and Y1767C mutations were transferred to the QQQmutant background and the time course of cocaine block wasdetermined by applying 900 ms depolarizing pulses to _10 mV. Inthe absence of cocaine, the decay of the current is biexponentialreflecting the slow inactivation of the channels (see text).A, cocaine (25–250 mM) induces the characteristic accelerateddecay in the QQQ-I1756C mutant current. B, same protocol withthe QQQ-Y1767C double mutant indicates that this channel isrelatively insensitive to cocaine. C, the decay of the QQQ-I1756Ccurrent was fitted with the sum of two exponentials and theapparent blocking rates (1/tf) plotted versus the cocaineconcentration. The QQQ-I1756C mutant has a kon and koff of2.6 w 105

M_1 s_1 and 26.3 s_1, respectively, yielding a KD of 101 mM.

The data suggest a KD of > 600 mM for the QQQ-Y1767C mutant.

Figure 9. Cocaine block of Na+ current in excised patchesOutside-out patches were excised from oocytes expressing theQQQ mutant. The Na+ in the internal pipette solution wasincreased to 140 mM and the external Na+ was replaced withcholine to facilitate the measurement of outward current. A,macroscopic currents from a patch elicited by a depolarizing pulseto +40 mV for 100 ms before and after bath application of 5 and50 mM cocaine. (VH = _100 mV). B, the decay of the current wasfitted with the sum of two exponentials and the apparent blockingrate (1/tf) plotted versus cocaine concentration. The linearrelationship is consistent with a bimolecular interaction withassociation and dissociation rate constants of 6.8 w 106

M_1 s_1 and

97.8 s_1, respectively, yielding a KD of 14.3 mM. Data are themeans ± S.E.M. of six to nine determinations.

outward current (Fig. 9). During a 100 ms depolarization

to +40 mV the decay of the current is well fitted by a single

exponential with a time constant of 49.0 ± 5.1 ms (n = 5).

Application of 5 and 50 mM cocaine induced a rapid

component of decay with time constants of 8.2 ± 1.6 ms

(n = 4) and 2.2 ± 0.1 ms (n = 5). This concentration-

dependent increase in blocking kinetics is consistent

with that previously observed in whole-oocyte recordings

(Fig. 5B). The time course of the decay was fitted with the

sum of two exponentials and the apparent blocking rates

(1/tf) plotted versus the cocaine concentration. As expected,

the rate of the cocaine block increased linearly with

concentration, with a KD of 14 mM. The estimate of cocaine

blocking affinity at +40 mV was nearly 10-fold greater

than that determined at _10 mV (KD = 122 mM) indicating

that strong depolarization further enhanced cocaine

binding. The midpoint and voltage sensitivity of the

activation of the QQQ mutant were _41.0 ± 0.4 mV and

5.7 ± 0.4 mV (n = 6), indicating that the channels were

maximally activated at both _10 and +40 mV. The

enhanced cocaine block cannot be attributed to differences

in the open probability at the more depolarized voltage.

One possible contributing factor to this apparent increase

in blocking affinity is that in addition to applying strong

depolarization, the external [Na+] had been reduced to

facilitate the measurement of outward currents. At more

hyperpolarized voltages (_25 mV), external Na+ and

cocaine appear to compete for binding sites (Fig. 6) and

reducing external Na+ is predicted to enhance the cocaine

block. We do not believe that the reduction in external [Na+]

can account for the increase in cocaine binding observed

in these experiments because previous studies have shown

that the Na+ antagonism of tetra-alkylammonium (TAA)

pore blockers is reduced or eliminated by strong depolariz-

ation (O’Leary et al. 1994). In effect, strong depolarization

appears to drive external Na+ out of the pore, thus

weakening its effect on TAA binding. Although we cannot

rule out a small contribution of external Na+ on the cocaine

binding, the entry of the positively charged cocaine into

the membrane electric field is likely to assume increased

importance when strong depolarizing test pulses are

applied.

Figure 10 shows an example of the effects of cocaine at the

single channel level. In the absence of the drug, the channels

were predominately open during the voltage pulse, resulting

in a slow decay of the ensemble average current. Cocaine

shortened the open times and induced a characteristic

decay in the ensemble average current at +40 mV similar

to our previously observations in macropatch experiments.

The mean open times before and after application of

cocaine were 5.2 and 1.2 ms, respectively. Cocaine reduced

the open times but did not alter the single-channel current

amplitude (2.4 pA), consistent with a discrete open-

channel blocking mechanism.

M. E. O’ Leary and M. Chahine710 J. Physiol. 541.3

Figure 10. Cocaine reduces the single-channel open timesRecordings of a single QQQ mutant channelbefore and after application of 10 mM cocaine.Recording configuration is identical to thatdescribed in Fig. 9. Currents were elicited by adepolarizing voltage pulse to +40 mV from aholding potential of _100 mV. Upward deflectionsrepresent channel openings. Cocaine (10 mM)reduced the mean open times but did not alter thesingle channel current amplitude (see text).Bottom, ensemble average open probabilityconstructed from 300 depolarizations for thecontrol and from 334 depolarizations in thepresence of cocaine. Calibration bars are 10 msand 2 pA.

DISCUSSION The inhibition of voltage-gated Na+ channels by local

anaesthetics is generally enhanced by depolarization and is

often associated with hyperpolarizing shifts in steady-state

inactivation, use-dependent inhibition and a delay in the

recovery of the channels at hyperpolarized voltages (Bean

et al. 1983; Sanchez-Chapula et al. 1983). The modulated

receptor hypothesis interprets such findings to reflect the

high-affinity binding of local anaesthetics to the open and

inactivated but not the closed states of Na+ channels (Hille,

1977; Hondeghem & Katzung, 1977). In this model,

charged forms of the anaesthetics are believed to bind

rapidly from the cytoplasmic side when the channels are

open. A second hydrophobic pathway has been proposed

that allows neutral forms of these anaesthetics to bind to

closed channels and that permits the drugs to escape when

the activation or inactivation gates are shut. More recent

evidence suggests that permanently charged quaternary

anaesthetics can also gain access to the internal binding site

by passing through the external mouth of the channel

(Alpert et al. 1989; Qu et al. 1995; Lee et al. 2001). The role

of these pathways in cocaine binding to and inhibition of

cardiac Na+ channels has not been determined.

Cocaine produces a use- and voltage-dependent inhibition

that is generally consistent with the modulated receptor

model. To investigate further the state-dependence of

cocaine binding, depolarizing prepulses were used to

stabilize the channels in the high-affinity inactivated

conformation (Fig. 2). Although the channels rapidly

occupy the inactivated state at depolarized voltages

(t ∆ 5 ms), the binding of cocaine is a comparatively slow

process. The reason for the slow time course of cocaine

binding is not known; however, this observation is

consistent with the proposed role of the inactivation gate,

which is believed to occlude the inner mouth of the

channel thus preventing cationic drugs such as cocaine

from rapidly accessing the binding site located within the

internal vestibule. This slow binding to inactivated channels

sharply contrasts with the rapid cocaine block of the QQQ

mutant (t ∆ 10 ms) where the drug gains access to the

binding site of open channels via the aqueous pathway. As

opposed to the open channels, direct cocaine binding to

inactivated channels may occur via the inherently slower

hydrophobic pathway. The cocaine inhibition of the

inactivated channels increases in a concentration-dependent

fashion with an apparent KD of 3.4 mM, similar to that

reported for heterologously expressed Nav1.5 (10 mM) and

native cardiac (8 mM) Na+ currents (Crumb & Clarkson,

1990; Wright et al. 1997). Mutations of the interdomain

III–IV linker that remove inactivation (IFMåQQQ)

abolish this high-affinity component of cocaine inhibition,

which appears to be linked to the fast inactivation of the

channel. This finding is in good agreement with previous

studies showing that mutations, neurotoxins, proteolytic

enzymes and chemical reagents that modify rapid

inactivation weaken or abolish local anaesthetic inhibition

(Cahalan, 1978; Yeh, 1978; Moczydlowski, 1986; Wang etal. 1987; Wasserstrom et al. 1993; Gingrich et al. 1993;

Bennett et al. 1995). Overall, the data provide strong

support for an important role for fast inactivation in the

high-affinity binding and are in good agreement with

previous studies of the cocaine inhibition of Na+ channels

(Wright et al. 1997, 1999).

A rapid component of cocaine inhibition persisted in

the inactivation-deficient (QQQ) mutant channels. At

concentrations < 100 mM, cocaine did not significantly

alter the peak amplitude but produced a pronounced,

time-dependent decay in the otherwise slowly inactivating

current (Fig. 5). The majority of the cocaine inhibition

developed after the channels had opened, with little tonic

block of the resting channels. The kinetics of the cocaine

inhibition of the QQQ mutant increased with concentration,

with an apparent KD at _10 mV of 122 mM. Depolarizing to

+40 mV further enhanced the cocaine block (KD = 14 mM),

suggesting that the binding site may be located within the

membrane electric field, similar to that reported for

cocaine (Wang, 1988) and pore blockers of Na+ channels

(O’Leary & Horn, 1994).

Reducing the external concentration of Na+ by 75 %

accelerated the cocaine-induced current decay, increased

the steady-state inhibition and caused a fivefold increase in

the affinity of cocaine block of the QQQ mutant (Fig. 6).

The data suggest that reducing the relative occupancy of

the pore by permeant cations facilitates cocaine binding.

Similar increases in blocking kinetics and affinity due to

changes in the concentration of external Na+ have been

observed for cocaine (Wang, 1988) and other compounds

that are known to inhibit Na+ channels by blocking the

pore (Cahalan & Almers, 1979; Gingrich et al. 1993;

O’Leary et al. 1994). The antagonism between cocaine

and external Na+ can result from either a competitive

interaction in which the blocker and Na+ compete for a

common site, or to a mechanism in which Na+ binds to a

separate site and indirectly destabilizes cocaine binding. In

principal, these mechanisms can be distinguished because

only the model incorporating two distinct binding sites

predicts a change in the dissociation rate constant for

cocaine binding (Wang, 1988). The data show that lowering

external Na+ decreased koff and increased kon (Fig. 6C) and

therefore favour a two-site model in which Na+ and

cocaine occupy adjacent binding sites within the pore.

When occupied, Na+ ions may destabilize cocaine binding

by a knock-off mechanism similar to that proposed for

other channel blockers (Armstrong, 1971; Cahalan &

Shapiro, 1980).

Overall, the high dependence of the cocaine inhibition on

channel opening, the voltage dependence of the block,

and the sensitivity to changes in the concentration of

Cocaine inhibition of Nav1.5 Na+ channelsJ. Physiol. 541.3 711

external Na+ are consistent with an open-channel blocking

mechanism for the QQQ mutant. This conclusion is further

supported by single channel measurements showing that

cocaine reduces the mean open times but does not alter the

current amplitude, properties that are characteristic of a

discrete open-channel blocker. At +40 mV, the open-

channel block and the binding of cocaine to inactivated

channels had similar affinities and both mechanisms are

likely to contribute to the cocaine inhibition of Nav1.5 Na+

channels.

Repriming of cocaine-modified channelsUse-dependent inhibition is a well-known property of

many local anaesthetics and is believed to result from the

preferential binding of the drugs to the open or inactivated

states of the channel. The use-dependence arises because

the drug-modified channels only slowly recover at hyper-

polarized voltages. The mechanism underlying this slow

repriming is a matter of considerable speculation. One

possibility is that the delay in repriming may reflect the

slow dissociation of the anaesthetic from the inactivated

channels (Courtney, 1981). The D4S6 mutations F1760C

and Y1767C accelerated the repriming of the drug-

modified channels and weakened the use-dependent

inhibition. Despite applying a high concentration of cocaine

(250 mM) that clearly inhibited the mutant channels, the

repriming of the cocaine-modified F1760C and Y1767C

channels remained rapid by comparison to the wild-type

(Fig. 4). The F1760C and Y1767C mutations did not

substantially alter the recovery of the drug-free channels,

indicating that this more rapid repriming does not result

from a non-specific effect of the mutations on the stability

of the inactivated state. The D4S6 mutations appear to

weaken the inhibition by promoting the more rapid

dissociation of cocaine when the cells are held at a

hyperpolarized voltage.

Removing fast inactivation by mutating the interdomain

III–IV linker (IFMåQQQ) reduced, but did not abolish,

the use-dependent inhibition. The data are consistent with

an important role for fast inactivation in the use-

dependent inhibition of the wild-type channel. The

residual use-dependent inhibition observed in the non-

inactivating mutant results from the cocaine block of open

channels which only slowly reprime at hyperpolarized

voltages. The rate constants for cocaine unbinding from

open channels were 57.9 s_1 at _10 mV and 66.3 s_1 at

_25 mV. Although somewhat limited in voltage range,

these estimates are consistent with previous studies

indicating that the rate of cocaine unbinding from open

channels increases with hyperpolarization (Wang, 1988).

Assuming that at low doses cocaine does not appreciably

bind to closed channels, the recovery time course of the

QQQ mutant at _100 mV yields an estimate of the

dissociation rate of 0.56 s_1 (Fig. 7B). The dissociation of

cocaine from closed channels is considerably slower

than that predicted from the kinetics and voltage

sensitivity of the open-channel block. The data indicate

that conformational changes that occur as the channels

deactivate stabilizes cocaine binding.

One possibility is that the affinity of cocaine binding may

increase as the channels deactivate. However, the binding

of cocaine to closed channels is generally weak by

comparison to that of open channels and such an increase

in binding affinity is inconsistent with most observations

of local anaesthetic inhibition. Alternatively, cocaine may

become trapped within the pore as the channels close, an

interpretation that is consistent with the open-channel

blocking mechanism. Closing of the activation gate

appears to prevent the rapid escape of cocaine from the

pore via the aqueous pathway. In order for the trapped

drug to dissociate from the binding site the activation gate

may have to reopen or cocaine convert to its neutral form

by losing a proton, thereby allowing the drug to exit the

pore by the hydrophobic pathway. This latter mechanism

may account for the unusually slow rate of repriming of

the cocaine-modified QQQ mutant channels at _100 mV

where the probability of channel opening is low. Similar

findings have been reported for the inhibition of Nav1.5

Na+ channels by a novel antiarrhythmic drug, suggesting

that pore block and untrapping may be a general

mechanism underlying the inhibition produced by many

anaesthetics (Pugsley & Goldin, 1999).

The repriming of the cocaine-modified wild-type channels

was biphasic with intermediate and slow time constants of

1.0 and 7.5 s, respectively. The intermediate component of

recovery measured in the wild-type had a similar time

constant as the untrapping of cocaine from the QQQ mutant

(t = 1.8 s), and may occur by a similar mechanism.

However, the majority of the wild-type channels recovered

with a time constant that was considerably slower than

that of untrapping, indicating additional mechanisms

contribute to the repriming. This slow component of

repriming was selectively eliminated by the interdomain

III–IV linker mutations that removes fast inactivation. The

data suggest that conformational changes associated with

fast inactivation further stabilize cocaine binding to its site

within the internal vestibule. An increase in cocaine binding

as the channels inactivate is predicted by the modulated

receptor model and is consistent with recent studies of

cocaine inhibition of Na+ channels (Wright et al. 1998).

Role of the D4S6 segment in cocaine binding to openand inactivated channelsResidues of the D4S6 segment of Na+ channels are known

to contribute to the binding of local anaesthetics. In rat

brain Na+ channels, mutations within the D4S6 segment

reduce the drug-induced use-dependent inhibition and

hyperpolarizing shift in steady-state inactivation (Ragsdale

et al. 1994). Mutation of the homologous residues in skeletal

muscle and cardiac Na+ channels produce similar effects

M. E. O’ Leary and M. Chahine712 J. Physiol. 541.3

on the binding of local anaesthetics and antiarrhythmic

drugs, indicating that the D4S6 binding site is conserved in

these Na+ channel isoforms (Wright et al. 1998; Nau et al.1999). Consistent with these previous studies, mutations

of highly conserved aromatic residues of the D4S6 segment

(F1760C, Y1767C) weakened the use-dependent inhibition,

slowed the onset and reduced the steady-state cocaine

inhibition of inactivated channels, and accelerated the

repriming of drug-modified channels. These effects on

cocaine inhibition do not appear to result from non-specific

changes in channel properties since these mutations did

not substantially alter the kinetics of inactivation or the

recovery of the drug-free channels. The data indicate that

the F1760C and Y1767C mutations primarily reduce the

cocaine inhibition by promoting the rapid dissociation of

the drug. The data are consistent with an important role

for these residues in cocaine binding to inactivated channels.

We have extended these findings by examining the effects

of these same D4S6 mutations on the cocaine inhibition of

the inactivation-deficient channels. The Y1767C mutation

abolished the open-channel block of the non-inactivating

mutant. These data are consistent with a recent study

showing that mutation of the F1760 homologue of the

skeletal muscle isoform weakens the discrete open-channel

block by QX-314 (Kimbrough & Gingrich, 2000). The data

indicate that residues of the D4S6 segment contribute to a

site that is important for cocaine binding in both the open

and inactivated conformations. According to the modulated

receptor hypothesis, local anaesthetics are believed to bind

to a single, high-affinity site located on the cytoplasmic side

of the channel (Hille, 1977). When this site is occupied, the

channel is unable to conduct current (i.e. blocked) and this

non-conducting conformation is further stabilized as the

channels inactivate. Our Y1767C and QQQ-Y1767C data

are in good agreement with these predictions.

Comparison with previous studies of localanaesthetic inhibitionA recent study suggested that recovery from fast-inactivated

states may not be the rate-limiting step in the repriming

of lidocaine-modified Na+ channels (Vedantham &

Cannon, 1999). This has led to the speculation that other

mechanisms, such as altered movement between activated

states, may account for the slow time course of repriming.

This has important implications since the high-affinity

binding to inactivated channels is believed to be a common

mechanism for many local anaesthetics, including cocaine.

However, in the study by Vedantham & Cannon (1998) high

concentrations of lidocaine were employed (0.1–8 mM),

which have previously been shown to block open channels

(Bennett et al. 1995). In addition, a mutation of the skeletal

muscle Na+ channel (Nav1.4: F1304C) was employed that

is known to destabilize fast inactivation (Vedantham &

Cannon, 1998). Our observation that the untrapping of

tertiary amine anaesthetics from closed channels is slow

may account for the finding that although the F1304C

mutant channels rapidly recover from inactivation they

only slowly recover from lidocaine block (Vedantham &

Cannon, 1998). In support of this idea, we have observed

that the untrapping of lidocaine from the QQQ mutant

channels (t ∆ 75 ms) has a time course similar to that

reported for the repriming of the lidocaine-modified

F1304C mutant channels (Vedantham & Cannon, 1999).

Our data suggest that the untrapping of lidocaine from

closed channels may, at least partially, explain the temporal

discrepancy between the rapid recovery from inactivation

and the slow repriming of drug-modified channels observed

in this previous study.

In wild-type channels, the onset of the cocaine inhibition

was slow by comparison to fast inactivation (t ∆ 5 ms) but

considerably more rapid than the development of slow

inactivation (t = 6.5 s). Clearly, the onset of the cocaine

inhibition is not well correlated with the availability of

either the fast or the slow inactivated states. Similar results

have been reported for the inhibition of Na+ channels by

lidocaine (Bennett et al. 1995). This discrepancy has

spawned suggestions that inactivated states with kinetics

intermediate between fast and slow inactivation may

contribute to anaesthetic binding (Kambouris et al. 1998).

We did observe a component of cocaine inhibition that has

similar intermediate kinetics. The time constants for this

inhibition were highly dependent on the drug concentration,

ranging from 1254 to 267 ms for 5 and 250 mM cocaine,

respectively. However, even at the highest concentration

(250 mM) the onset of the cocaine inhibition of the wild-

type channels was considerably slower than that reported

for the development of intermediate slow inactivation

(t = 70 ms; Veldkamp et al. 2000). The concentration-

dependent increase in cocaine inhibition was not well

correlated with the development of intermediate slow

inactivation. In addition, the majority of the cocaine

inhibition measured at _10 mV was abolished by mutations

of the interdomain III–IV linker that remove fast

inactivation, and the residual cocaine inhibition of the

non-inactivating mutant can be attributed to an open-

channel blocking mechanism. Overall, the data do not

provide strong support for an important role for

intermediate and slow inactivation in the cocaine inhibition

of Nav1.5 Na+ channels.

Our data indicate that the open and fast-inactivated states

of Nav1.5 are the most important for cocaine binding.

Although the channel rapidly adopts these high-affinity

configurations at depolarized voltages, the onset of the

cocaine inhibition is comparatively slow. Inactivation

appears to prevent the rapid binding of cocaine via the

aqueous cytoplasmic pathway. The slow onset of the cocaine

inhibition during depolarizations > 10 ms in duration is

likely to reflect the poor accessibility of the drug to the

internal binding site of inactivated channels. When the

Cocaine inhibition of Nav1.5 Na+ channelsJ. Physiol. 541.3 713

channels are inactivated, cocaine may have to convert to

the neutral form before accessing the binding site via a

hydrophobic pathway, which appears to be considerably

slower than the direct binding of the charged drug via the

aqueous cytoplasmic pathway. This slower binding may

account for the poor temporal correlation between the

development of the high-affinity conformation that is linked

to fast inactivation and the onset of cocaine inhibition.

A recent study of the cardiac Na+ channel suggested that

the direct access of anaesthetics via an external pathway

may be equivalent to or, in some cases, more important

than drug binding through the cytoplasmic pathway (Lee

et al. 2001). Although an external pathway for cocaine

permeation cannot be ruled out, we feel that cocaine

primarily accesses the binding site from the cytoplasmic

side of the channel for several reasons. Cocaine inhibition

at concentrations < 100 mM occurred predominately after

the channels had opened, suggesting that the drug may be

unable to gain access when the channels are closed and

deactivation traps cocaine within the pore. The data

indicate that when closed, the cytoplasmic activation gate

acts as an effective barrier that slows the binding and

unbinding of cocaine to the D4S6 site. The binding of

cocaine was voltage dependent, increasing with depolariz-

ation, suggesting that the drug enters the membrane

electric field to reach its binding site. Cocaine is positively

charged at physiological pH, indicating that the cationic

drug must enter the membrane electric field from the

cytoplasmic side of the channel. The block produced by a

cationic drug entering the electric field from the external side

of the channel would be weakened by strong depolariz-

ation. Finally, the binding of cocaine to the open channel

was antagonized by raising the external concentration Na+,

consistent with a model in which cocaine and Na+ bind to

distinct, but functionally overlapping, sites within the pore.

It seems likely that the effect of raising the concentration of

external Na+ on drug approaching the binding site from

the outside of the channel might manifest as a competitive

mechanism, rather than the non-competitive mechanism

observed in these studies. Overall, the data support a

model in which cocaine blocks the closed and open

channels by approaching through the aqueous cytoplasmic

pathway. We currently do not have a thorough under-

standing of the access pathway used by cocaine when the

channels are inactivated. An external pathway similar to

that described by Lee et al. (2001) may be important for

cocaine binding under these conditions.

Mechanism of cocaine inhibitionTwo distinct components of cocaine inhibition were

identified in the wild-type and inactivation-deficient

mutant Nav1.5 Na+ channels. A high-affinity component

of inhibition with slow onset kinetics was observed in the

wild-type channels that appeared to be linked to fast

inactivation. A second component of cocaine inhibition

was observed in the inactivation-deficient mutant channel

that had rapid kinetics and displayed properties that

are consistent with a simple pore-blocking mechanism.

The relationship between the open-channel block and

inactivation-dependent components of cocaine inhibition

is currently unclear. However, Nav1.5 Na+ channels only

briefly open (< 1 ms) in response to depolarizing voltage

pulses (O’Leary & Horn, 1994). Considering the relatively

slow kinetics of cocaine binding to the open channels

(t = 3–16 ms), only a very small fraction of the activated

channels are expected to bind cocaine before inactivating.

The majority of the channels rapidly inactivate and bind

cocaine at a considerably slower rate. However, in the

small fraction of blocked channels the binding of cocaine is

likely to be further stabilized by inactivation. During rapid

repetitive stimulation, the combination of pore block and

inactivation would tend to cause a progressive increase in

the number of drug-modified channels at the end of each

voltage pulse. These channels would be unable to fully

recover until the repetitive stimulation was terminated and

the membrane potential was returned to a hyperpolarized

voltage for a prolonged interval (> 10 s). Our data suggest

that the rapid block of open channels, coupled with the

increased affinity that occurs as the channels inactivate

may act cooperatively to produce the cocaine inhibition of

cardiac Na+ channels observed during rapid repetitive

stimulation.

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AcknowledgementsWe would thank Dr Richard Horn for helpful comments on themanuscript. This work was supported by grants from the AmericanHeart Association (9730216N) and the National Institute on DrugAbuse (DA15192).

M. E. O’ Leary and M. Chahine716 J. Physiol. 541.3