Journal of Physiology (2002), 541.3, pp. 701–716 © The Physiological Society 2002
DOI: 10.1113/jphysiol.2001.016139 www.jphysiol.org
Cocaine binds to a common site on open and inactivated human heart (Nav1.5) sodium channels M. E. O’Leary and M. Chahine Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA, USA and Quebec Heart Institute, Laval Hospital Research 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, 1020 Locust Street JAH 266, Philadelphia, PA 19107, USA. Email: michael.o’
[email protected]
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).
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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 inactivationdeficient mutant that had properties consistent with a pore-blocking mechanism. Mutations of the D4S6 segment weakened both the inactivation-dependent and poreblocking components, consistent with a common, highaffinity binding site for cocaine located within the internal vestibule of the channel.
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METHODS Site-directed mutagenesis Amino acid substitutions were made using the QuickChange SiteDirected Mutagenesis Kit (Stratagene Inc., La Jolla, CA, USA). Fragments (1–2 kb) encompassing the mutation sites were excised and subcloned into pcDNA. Complementary pairs of oligonucleotides (22–29 bp) containing the appropriate nucleotide substitutions were prepared at the Nucleic Acid Facility, Kimmel Cancer Center (Thomas Jefferson University, Philadelphia, PA, USA). These oligonucleotides were subsequently used as primers for the complete synthesis of both strands of the plasmid. We used 20 ng of cDNA plasmid as template, 5 U Pfu DNA polymerase (Stratagene, Inc.), primers, and free nucleotides in a total volume of 100 ml. After strand synthesis (~20 cycles), 10 U of Dpn I was added to the reaction mixture to digest the original methylated plasmid template (37 °C, 1–2 h). The restriction endonuclease was heat inactivated (65 °C, 15 min), and the mixture used to transform DH5a cells (Gibco BRL) by electroporation. Base substitutions were confirmed by automated DNA sequencing by the Nucleic Acid Facility of the Kimmel Cancer Center at Jefferson Medical College. DNA fragments (1–2 kb) carrying the mutation were then sub-cloned into wild-type Nav1.5 background and amplified in DH5a. Oocyte expression and two-electrode voltage clamp The cDNA encoding the human cardiac (Nav1.5) Na+ channel in the pcDNA plasmid (Invitrogen) was linearized with Xba I and full length capped mRNA transcribed using the T7 promoter (mMessage mMachine, Ambion, Austin, TX, USA). Oocytes were harvested from mature female Xenopus laevis (Xenopus I, Ann Arbor, MI, USA). The animals were anaesthetized by immersion in tricaine (1.5 mg ml_1) and several ovarian lobes surgically removed under sterile conditions. The adhering follicle cell layer was 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-free OR2 and transferred to 70 % Leibovitz L-15 medium (Life Technologies) supplemented with 15 mM Hepes (pH 7.4), 5 mM _1 L-glutamine and 10 mg ml gentamycin. Stage IV–V oocytes were microinjected with 50 nl of cRNA (1–2 mg ml_1) and incubated for 24–48 h at 18 °C. The animals were treated in accordance with the NIH guidelines and the protocol was approved by the Animal Use and Care Committee of Thomas Jefferson University. The currents of cRNA-injected oocytes were recorded using a standard two-electrode voltage clamp technique. Oocytes were impaled with microelectrodes (< 1 MV) filled with 3 M KCl and currents recorded using an OC-725C voltage clamp (Warner Instruments, Hamden, CT, USA). Oocytes were held at _100 mV and pulses generated using pCLAMP software (Version 7, Axon Instruments, Foster City, CA, USA). ND96 recording saline contained (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 recordings Excised-patch recordings were obtained using an Axopatch 200A patch clamp amplifier equipped with a DigiData 1200 interface (Axon Instruments). Voltage pulses were generated and data collected using pCLAMP (Version 7, Axon Instruments). Patch pipettes were fashioned from Corning 8161 glass (Dow Corning) with resistances of 0.5–2 MV and were Sylgard coated to reduce
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capacity transients. Extracellular solution contained (mM): 140 choline chloride, 2 KCl, 1.5 CaCl2, 1 MgCl2 and 10 Hepes, pH 7.4 with KOH. Pipette solution consisted of (mM): 140 NaCl, 5 EGTA and 10 Hepes, pH 7.4 with NaOH. Patches were held at _100 mV and 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 by isosmotically replacing NaCl with choline chloride. Cocaine-HCl was purchased from the Jefferson Hospital Pharmacy and prepared as a 50 mM stock solution in water.
RESULTS Cocaine produced a use-dependent inhibition of Nav1.5 Na+ channels At 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 et al. 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.
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Time course of the cocaine inhibition Although 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
Figure 1. Use-dependent inhibition of Nav1.5 Na+ channels expressed in Xenopus oocytes The use-dependent inhibition was induced by a series of 50 depolarizing pulses to _10 mV for 20 ms applied at a frequency of 5 Hz. A and B, currents of the wild-type (A) and F1760C mutant (B) after bath application of 50 mM cocaine. Current traces correspond to the pulse numbers 1, 5, 10, and 50 of the stimulation train. C–F, the peak current elicited by each pulse within the train was normalized to the current of the first pulse and plotted versus the 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).
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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.
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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 steadystate 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).
Figure 2. Time course of cocaine binding to inactivated channels A, the time course of cocaine inhibition was measured using a triple pulse protocol consisting of a conditioning pulse to _10 mV of variable duration (1 ms–60 s), a short hyperpolarization to _100 mV for 150 ms and a test pulse to _10 mV. the peak currents elicited by test pulses were normalized to controls (I/Io) and plotted versus the prepulse duration. The decay of the current is best described by the sum of two exponentials : I/Io = A1exp(_t/ t1) + A2 exp(_t/ tS) + AW, where A1, A2 and AW are the relative amplitudes of the fast, slow and steady 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 and 3494 ± 1592 ms for 50 mM (n = 6), 6 ± 4 and 581 ± 21 ms for 100 mM (n = 11), 8 ± 6 ms and 267 ± 25 ms (n = 5) for 250 mM cocaine. B, time constants of the fast, intermediate, and slow components of current decay plotted versus the cocaine concentration (see text). C, plot of the normalized steady-state current amplitude (AW) versus the cocaine concentration. The smooth curve is a fit to a single site model with a KD of 3.4 ± 0.4 mM.
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) versus the concentration (Fig. 2C). Cocaine reduced the steadystate 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
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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 steadystate 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.
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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
Repriming of cocaine-modified channels The 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
Figure 3. Effects of D4S6 mutations on cocaine inhibition The onset of cocaine inhibition was measured using the same pulsing protocol as in Fig. 2. In the absence of cocaine, the development of slow inactivation was biexponential with fast and slow 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 the time 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). The fraction of non-inhibited current (AW) before and after addition of 50 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 and 0.43 ± 0.02 for Y1767C. Also plotted is the inhibition of the F1760C and Y1767C mutants by 250 mM cocaine with fast and slow time 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).
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Table 1. Time course of the recovery from inactivation and cocaine inhibition t1 (ms)
t2 (ms)
t3 (ms)
A1
A2
A3
Wild-type Control 50 mM
11 ± 2 11 ± 2
189 ± 150 1017± 327
1967 ± 872 7497 ± 560
0.58 ± 0.04 0.17 ± 0.01
0.19 ± 0.06 0.19 ± 0.04
0.23 ± 0.07 0.65 ± 0.04
I1756C Control 50 mM
12 ± 2 23 ± 8
189 ± 103 1190 ± 339
3074 ± 1168 7882 ± 1202
0.52 ± 0.05 0.21 ± 0.04
0.25 ± 0.05 0.33 ± 0.07
0.23 ± 0.05 0.47 ± 0.07
F1760C Control 50 mM 250 mM
5±1 4±1 10 ± 3
255 ± 80 242 ± 70 348 ± 123
2363 ± 1024 2498 ± 361 2569 ± 179
0.41 ± 0.03 0.34 ± 0.02 0.11 ± 0.01
0.37 ± 0.06 0.25 ± 0.04 0.18 ± 0.04
0.22 ± 0.07 0.41 ± 0.04 0.71 ± 0.04
Y1767C Control 50 mM 250 mM
4±1 3±1 10 ± 3
118 ± 99 76 ± 62 343 ± 65
1395 ± 327 1121 ± 293 4350 ± 1013
0.46 ± 0.04 0.40 ± 0.07 0.26 ± 0.03
0.16 ± 0.06 0.19 ± 0.07 0.46 ± 0.04
0.39 ± 0.06 0.41 ± 0.07 0.27 ± 0.04
The time constants (t) and relatively amplitudes (A) were determined from fits of the recovery data to the sum of three exponentials (Fig. 4). The data are shown before (Control) and after bath application of 50 or 250 mM cocaine. The data are the means ± standard errors. Wild-type, n = 7; I1756C, n = 3; F1760C, n = 8; Y1767C, n = 9.
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 drugmodified channels.
Cocaine inhibition of inactivation-deficient channels The contribution of fast inactivation to the cocaine inhibition was further investigated using an inactivationdeficient 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-
Figure 4. Time course of recovery from cocaine inhibition Channels were depolarized for 10 s to _10 mV before returning to _100 mV for intervals ranging from 1 ms to 30 s. A _10 mV test pulse was then used to assay the fraction of recovered current, which was normalized to control test currents and plotted versus the recovery interval. The recovery 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 of three exponentials with the parameters listed in Table 1.
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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 105 M_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).
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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 hyperpolarization 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 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 timedependent 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 fast inactivation Similar 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).
Figure 5. The time course of cocaine block of the QQQ mutant channels The inactivation-deficient mutant (IFMåQQQ) was constructed by replacing a series of hydrophobic residues of the interdomain III–IV linker with glutamines (see text). A, currents were elicited by depolarizing for 2 s to _10 mV from a holding potential of _100 mV. In the absence of cocaine, the decay of the current is biexponential with time constants of 85.6 ± 6.2 and 1063.4 ± 98.4 ms (n = 6). After application of 50 mM cocaine the decay of the current was fitted with the sum of three exponentials with time constants of 15.2 ± 0.6, 181.5 ± 7.0 and 1519.3 ± 85.7 ms (n = 6). The cocaine inhibition was completely reversible upon washout with time constants of 75.4 ± 6.8 and 1154.6 ± 180.5 ms (n = 6). B, the time course of cocaine inhibition during 200 ms depolarizations was measured over a range of concentrations and the decay time constants determined from biexponential 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.
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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
Figure 6. Cocaine block is sensitive to changes in the concentration of external Na+ A, whole-cell currents of the QQQ mutant measured in normal Ringer solution (116 mM Na+) before and after the application 100 mM cocaine. Currents were activated by depolarizing to _25 mV from a holding potential of _100 mV. B, same pulsing protocol was applied in reduced external Na+ Ringer solution (29 mM Na+). C, the blocking rates were determined from the rapid decay of the currents as described previously. The association and dissociation 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 105 M_1 s_1 and 27.3 s_1 for the 25 % Na+ conditions. The KD of cocaine inhibition at _25 mV is 214 mM in the high external Na+ and 43 mM in low external Na+ Ringer solution.
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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 of non-inactivating channels The 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
Figure 7. Use-dependent inhibition of the QQQ mutant Na+ channels A, a series of 50 depolarizing pulses to _10 mV for 20 ms were applied at a frequency of 5 Hz. Currents elicited by individual pulses were normalized and plotted versus pulse number. The usedependent 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-type channel in 100 % Na+ solution replotted from Fig. 1C. B, repriming time course of cocaine-blocked channels. Cells were depolarized for 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 assay availability after the completion of the recovery interval. In the presence of cocaine the recovery time course is well fitted by a single exponential with a time constant of 1.8 ± 0.1 s and a steadystate amplitude of 0.43 ± 0.05 (n = 8). Also plotted is the time course of recovery from slow inactivation which has a time constant of 49.3 ± 5.0 ms and a steady-state current amplitude of 0.91 ± 0.01 (n = 9).
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Cocaine inhibition of Nav1.5 Na+ channels
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.
Figure 8. Effects of D4S6 mutations on cocaine block The I1756C and Y1767C mutations were transferred to the QQQ mutant background and the time course of cocaine block was determined by applying 900 ms depolarizing pulses to _10 mV. In the absence of cocaine, the decay of the current is biexponential reflecting the slow inactivation of the channels (see text). A, cocaine (25–250 mM) induces the characteristic accelerated decay in the QQQ-I1756C mutant current. B, same protocol with the QQQ-Y1767C double mutant indicates that this channel is relatively insensitive to cocaine. C, the decay of the QQQ-I1756C current was fitted with the sum of two exponentials and the apparent blocking rates (1/tf) plotted versus the cocaine concentration. The QQQ-I1756C mutant has a kon and koff of 2.6 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.
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Voltage sensitivity of the cocaine inhibition of the QQQ mutant The 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 openchannel block in the cocaine inhibition of Na+ channels in vivo 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
Figure 9. Cocaine block of Na+ current in excised patches Outside-out patches were excised from oocytes expressing the QQQ mutant. The Na+ in the internal pipette solution was increased to 140 mM and the external Na+ was replaced with choline to facilitate the measurement of outward current. A, macroscopic currents from a patch elicited by a depolarizing pulse to +40 mV for 100 ms before and after bath application of 5 and 50 mM cocaine. (VH = _100 mV). B, the decay of the current was fitted with the sum of two exponentials and the apparent blocking rate (1/tf) plotted versus cocaine concentration. The linear relationship is consistent with a bimolecular interaction with association 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 the means ± S.E.M. of six to nine determinations.
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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 concentrationdependent 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
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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 depolarization (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 openchannel blocking mechanism.
Figure 10. Cocaine reduces the singlechannel open times Recordings of a single QQQ mutant channel before and after application of 10 mM cocaine. Recording configuration is identical to that described in Fig. 9. Currents were elicited by a depolarizing voltage pulse to +40 mV from a holding potential of _100 mV. Upward deflections represent channel openings. Cocaine (10 mM) reduced the mean open times but did not alter the single channel current amplitude (see text). Bottom, ensemble average open probability constructed from 300 depolarizations for the control and from 334 depolarizations in the presence of cocaine. Calibration bars are 10 ms and 2 pA.
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Cocaine inhibition of Nav1.5 Na+ channels
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
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enzymes and chemical reagents that modify rapid inactivation weaken or abolish local anaesthetic inhibition (Cahalan, 1978; Yeh, 1978; Moczydlowski, 1986; Wang et al. 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
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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 openchannel 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 channels Use-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 hyperpolarized 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 drugmodified 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 usedependent inhibition of the wild-type channel. The residual use-dependent inhibition observed in the noninactivating 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
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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 open and inactivated channels Residues 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
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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 local anaesthetic inhibition A 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
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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 wildtype channels was considerably slower than that reported for the development of intermediate slow inactivation (t = 70 ms; Veldkamp et al. 2000). The concentrationdependent 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 openchannel 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
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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 depolarization, 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 depolarization. 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 understanding 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 inhibition Two 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
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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.
REFERENCES ALPERT, L. A., FOZZARD, H. A., HANCK, D. A. & MAKIELSKI, J. C. (1989). Is there a second external lidocaine binding site on mammalian cardiac cells? American Journal of Physiology 257, H79–84. ARMSTRONG, C. M. (1971). Interaction of tetraethylammonium ion derivatives with the potassium channel of giant axon. Journal of General Physiology 58, 413–437. ARMSTRONG, C. M., BEZANILLA, F. & ROJAS, E. (1973). Destruction of sodium conductance inactivation in squid axons perfused with pronase. Journal of General Physiology 62, 375–391. BEAN, B. P., COHEN, C. J. & TSIEN, R. W. (1983). Lidocaine block of cardiac sodium channels. Journal of General Physiology 81, 613–642. BENNETT, P. B., VALENZUELA, C., CHEN, L. Q. & KALLEN, R. G. (1995). On the molecular nature of the lidocaine receptor of cardiac Na+ channels. Modification of block by alterations in the alpha-subunit III-IV interdomain. Circulation Research 77, 584–592. BILLMAN, G. E. (1995). Cocaine: a review of its toxic actions on cardiac function. Critical Review of Toxicology 25, 113–132. CAHALAN, M. D. (1978). Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons. Biophysical Journal 23, 285–311. CAHALAN, M. D. & ALMERS, W. (1979). Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin. Biophysical Journal 27, 39–55. CAHALAN, M. D. & SHAPIRO, B. A. W. (1980). Relationship between inactivation of sodium channels and block by quaternary derivatives of local anesthetics and other compounds. In Molecular Mechanisms of Anesthesia, ed. FINK, B. R., pp. 17–33. Raven Press, New York.
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Acknowledgements We would thank Dr Richard Horn for helpful comments on the manuscript. This work was supported by grants from the American Heart Association (9730216N) and the National Institute on Drug Abuse (DA15192).
