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Institute of Pharmacology, Medical University of Vienna, Vienna, Austria (W.S., J.S., T.Z., E.Z., K.H., H.T.); Cardiac Electrophysiology Laboratories, University of Chicago, Chicago, Illinois (I.G., H.A.F.); and Division of Cardiology, Emory University, Atlanta, Georgia and Atlanta Veterans Affairs Hospital, Decatur, Georgia (S.C.D.)
Received October 22, 2003; accepted June 9, 2004
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Abstract |
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rec = 171 ± 19 s), typical for recovery from IUS. After exposure to 300 µM lidocaine, the fraction of channels recovering from IUS was reduced to 13 ± 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on IUS, indicating that lidocaine has to bind to the inner vestibule of the channel to modulate IUS. We propose that IUS involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.
). Furthermore, certain mutations in the selectivity filter region promote an ultra-slow inactivated state (IUS). Entry into and exit from IUS requires up to 20 min (Todt et al., 1999; Hilber et al., 2001, 2002).
Whereas the molecular basis of IF is likely to be a "hinged lid" mechanism involving a cytoplasmic loop between the third and fourth domain [the III–IV linker, (West et al., 1992)], little is known about the mechanistic basis of the slower forms of inactivation.
It has been suggested that slower forms of inactivation occur by a closure of the extracellular mouth of the channel (Tomaselli et al., 1995; Balser et al., 1996; Townsend and Horn, 1997; Kambouris et al., 1998; Todt et al., 1999; Ong et al., 2000; Hilber et al., 2001, 2002; Vilin et al., 2001; Xiong et al., 2003).
Lysine 1237 is located in the outer vestibule of the rNaV 1.4 channel and has a pivotal role in ionic selectivity (Favre et al., 1996), suggesting that this residue is part of the selectivity filter (Lipkind and Fozzard, 1994). The mutation K1237E favors entry into IUS supporting the notion that IUS is produced by a conformational change of the outer vestibule. On the other hand, we recently found that IUS is inhibited by closure of the intracellular inactivation gate indicating that the intracellular vestibule may participate in the molecular motion that underlies IUS (Hilber et al., 2002).
To explore the latter notion, we sought to explore the effect of lidocaine on IUS. Lidocaine blocks voltage-gated Na+ channels by binding to the inner vestibule and by interaction with the inactivated conformation of the channel (Hille, 1977; Hondeghem and Katzung, 1977; Ragsdale et al., 1994). We examined the effect of lidocaine in the mutant K1237E because 100% of channels can be driven into IUS in this mutant, whereas in wild-type channels only 
20% of channels enter into IUS in response to long depolarizing pulses (Todt et al., 1999). We found that lidocaine dramatically increased the apparent time constant of entry into IUS whereas the time constant of recovery from IUS was unaffected. These data are consistent with a model in which IUS occurs by a closure of the inner vestibule. Lidocaine may enter the inner vestibule and inhibit the channel closure via a foot-in-the-door mechanism. Some of the data have been published in abstract form (Sandtner et al., 2004).
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Materials and Methods |
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).
Site-Directed Mutagenesis. Detailed methods for the mutagenesis have been published previously (Dudley et al., 1995; Sunami et al., 1997; Todt et al., 1999).
Electrophysiological Recordings. Stage V and VI Xenopus laevis oocytes were isolated from female frogs (Nasco, Ft. Atkinson, WI), washed with Ca2+-free solution (90 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM NaHPO4, and 5 mM HEPES titrated to pH 7.6 with 1 N NaOH), treated with 2 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO) for 1.5 h, and their follicular cell layers were manually removed. As judged from photometric measurements, approximately 50 to 100 ng of cRNA was injected into each oocyte with a Drummond micro-injector (Drummond Scientific Co., Broomall, PA). Either native or mutant 
subunit cRNA was mixed with rat brain 
1 cRNA at a molar ratio of 1:1. Oocytes were incubated at 17°C for 12 h to 3 days before examination.
Recordings were made in the two-electrode voltage clamp configuration using a TEC 10CD clamp (NPI Electronic GmbH, Tamm, Germany). For accurate adjustment of the experimental temperature (18 ± 0.5°C), an oocyte bath cooling system (HE 204; Dagan Corp., Minneapolis, MN) was used. Oocytes were placed in recording chambers in which the bath flow rate was about 100 ml/h, and the bath level was adjusted so that the total bath volume was less than 500 µl. Electrodes were filled with 3 M KCl and had resistances of less than 1 M
. Using pCLAMP6 software (Axon Instruments Inc., Foster City, CA), data were acquired at 71.4 kHz after low-pass filtration at 2 kHz (–3 db). Curve fitting was performed using ORIGIN 5.0 (OriginLab Corp., Northampton, MA). Recordings were made in a bathing solution that consisted of 90 mM NaCl, 2.5 mM KCl, 1 mM BaCl2, 1 mM MgCl2, and 5 mM HEPES titrated to pH 7.2 with 1 N NaOH. BaCl2 was used as a replacement for CaCl2 to minimize Ca2+-activated Cl– currents. Lidocaine was obtained from Sigma-Aldrich.
Data Evaluation. The normalized time courses of recovery from IUS of normalized peak inward currents were fit with the double exponential function:
 | (1) |
1 and 2 are the time constants of distinct components of recovery, A1 and A2 are the respective amplitudes of these components.
The time course of entry into IUS was fitted with the monoexponential function:
 | (2) |
Data are expressed as means ± S.E.M. Statistical comparisons were made using one-way analysis of variance. A P 
0.05 was considered significant.
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Results |
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100 s. We refer to this state as IUS (Todt et al., 1999). To test, whether exposure to lidocaine affects IUS, recovery from IUS was assessed in NaV 1.4 channels during a drug-free control and after exposure to 300 µM lidocaine (Fig. 1A). The membranes were depolarized to –20 mV for 300 s. Thereafter, the potential was returned to –120 mV, and the time course of recovery from inactivation was monitored by 20-ms test pulses to –20 mV applied at 20-s intervals. Under control conditions, recovery had a biexponential time course with 80% of channels recovering with a time constant of 6.5 s, reflecting recovery from slow inactivation, which is the predominant state from which wild-type channels recover in response to long depolarizations (Kambouris et al., 1998; Todt et al., 1999). Twenty percent of channels recovered with a time constant of 170 s, reflecting recovery from IUS. During lidocaine exposure, the fraction of channels recovering from IUS was almost completely removed. The small fraction of channels recovering from IUS in wild-type channels even under lidocaine-free conditions precluded a systematic analysis of this drug effect in wild-type channels.
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). Therefore, we examined the effect of lidocaine on IUS in the mutant K1237E. Figure 1B shows the effect of lidocaine in an oocyte-expressing K1237E channels. Recovery from IUS was examined as in Fig. 1A. Clearly, under control conditions most K1237E channels recovered with a very slow time course (Fig. 1B; arrow "recovery control"). After full recovery from IUS, 300 µM lidocaine were washed in, resulting in blockage of 17% of inward current. Because this block developed at –120 mV, it most likely reflects low-affinity binding of lidocaine to Na+ channels in the rested state ("tonic block"). Following the establishment of steady-state tonic block, the time course of recovery from IUS was assessed again. Lidocaine substantially speeded recovery from IUS (Fig. 1B, arrow "recovery lidocaine"). In Fig. 1C, the normalized time courses of recovery from IUS during exposure to lidocaine concentrations of 5 to 300 µM are presented. Fitting two exponentials (eq. 1) to the time courses of recovery revealed that lidocaine had no effect on the time constant of recovery from IUS (2) but decreased the fraction of channels recovering from IUS (A2), suggesting that lidocaine may have increased the time constant of entry into IUS without affecting the time constant of exit from this state (Table 1).
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Lidocaine Modulates IUS through a Binding Site in the Inner Vestibule. The residue Phe1579, which is located in the intracellular vestibule of the Na+ channel, forms a part of the binding site of local anesthetic drugs. Accordingly, the mutation F1579A reduced the binding affinity of open and inactivated channels for lidocaine by 2- and 24.5-fold, respectively (Ragsdale et al., 1996). We wondered whether the effect of lidocaine on IUS was mediated by binding of lidocaine to this site. Hence a double mutant carrying the mutations K1237E and F1579A was constructed. To confirm that the double mutant K1237E + F1579A has a reduced binding affinity for lidocaine, we tested the effect of 300 µM lidocaine on currents evoked by repetitive 15-ms test pulses at varying frequencies. As shown in Fig. 2A, the single mutant K1237E was inhibited by lidocaine in a frequency-dependent manner. Such frequency-dependent block is due to the transient availability of higher affinity channel states during each stimulus pulse. The high sensitivity of K1237E channels to frequency-dependent block by lidocaine indicates a high affinity of open and/or inactivated channels for lidocaine. By contrast, the frequency-dependent block was almost abolished in the double mutant K1237E + F1579A (Fig. 2A). Thus, the additional mutation F1579A effectively abolished lidocaine binding to the high-affinity states. Next we tested the effect of lidocaine on IUS, which takes several minutes to develop. Recovery from IUS was assessed as in Fig. 1. Lidocaine (300 µM) had only a slight effect on IUS in the double mutant K1237E + F1579A (Fig. 2B) when compared with the single mutant K1237E (Fig. 1). This suggests that lidocaine has to bind to the cytoplasmic vestibule of the channel to inhibit entry into IUS.
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Lidocaine Increases Occupancy of IM in the Mutant K1237E. In wild-type NaV 1.4 channels, one reported effect of lidocaine is to increase occupancy of IM (Kambouris et al., 1998; Chen et al., 2000; Ong et al., 2000). Given the dramatic effect of lidocaine on IUS in K1237E, we wondered whether the lidocaine-induced occupancy of IM is preserved in this mutant. To assess the development of binding of lidocaine to IM, oocytes expressing K1237E channels were exposed to 300 µM lidocaine. From a holding potential of –120 mV, channels were depolarized to –20 mV for 10 ms (Fig. 3, A and B, inset). Thereafter, the membrane potential was returned to –120 mV and the time course of recovery from inactivation was assessed by 50 ms test pulses to –20 mV applied after variable recovery intervals (Fig. 3A). Channels recovered with two time constants 1 = 2.2 ± 0.25 ms and 2 = 747 ± 252.9 ms, reflecting the time constants of recovery from fast inactivation (IF) and IM, respectively (Chen et al., 2000). The fractions of channels recovering from IF and from IM were 0.74 ± 0.04 and 0.26 ± 0.02, respectively (A1 and A2 in eq. 1)
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With longer prepulse durations, the fraction of channels recovering from IM (A2) increased substantially (0.76 ± 0.03 after a prepulse of 500 ms duration); however, prolonging the prepulse duration from 500 to 1000 ms did not result in any further substantial increase in the fraction of channels entering into the IM state. These data suggest that lidocaine increased the occupancy of IM and the development of this process was completed within about 500 ms. Thus, similar to wild-type NaV 1.4 channels (Chen et al., 2000), conditioning prepulses of 1000 ms duration result in steady-state occupancy of IM by lidocaine in K1237E.
Next we assessed the concentration dependence of the lidocaine-induced occupancy of IM. K1237E channels were depolarized for 1 s and recovery from inactivation was examined with a double pulse protocol. Under lidocaine-free conditions, full recovery of test pulse current was attained after 10 s (Fig. 3B). Lidocaine did not change the time to full recovery but produced a concentration-dependent decrease in the fraction of channels recovering from IF and a corresponding increase in the fraction of channels recovering from IM (Fig. 3C, Table 2). According to Khodorov et al. (1976) and Bean et al. (1983), the strength of lidocaine binding to an inactivated state can be deduced from the relative fraction of channels recovering from that state. We quantified the interaction of lidocaine with IM by calculating the fraction of channels unavailable to recover from IM (Fnon-IM) at a given concentration of lidocaine according to Fnon-IM = 1 – (A2-lido – A2-control)/(A2-max – A2-control), where A2-lido and A2-control are the fractions recovering from IM during exposure to lidocaine and during drug-free control, respectively. A2-max is the maximal fraction recovering from IM that was found during exposure to 1000 µM lidocaine. The apparent KD for the reduction in Fnon-IM produced by lidocaine was 22.1 ± 2.6 µM (Fig. 3D). These data show that K1237E retains the state-dependent lidocaine sensitivity typical for wild-type µ1 channels (Kambouris et al., 1998).
Lidocaine Does Not Alter the Distribution of Channels between IM and IS. As mentioned above, IS in Nav 1.4 channels is defined as an inactivated state with time constants of recovery on the order of several thousand milliseconds (Kambouris et al., 1998). To examine the effect of lidocaine on IS wild-type Nav 1.4 channels were depolarized for 25sto –20 mV, and the time course of recovery was assayed as shown in Fig. 3C. Because lidocaine has a substantial effect on IUS, we chose to evaluate the effect of lidocaine on IS in wild-type channels to minimize possible interfering effects of the drug on the neighboring IUS state. As shown in Fig. 4, the time course of recovery from a 25-s prepulse could be fit with a double exponential formula (eq. 1) yielding two time constants 1 = 932 ± 210 ms and 2 = 5620 ± 810 ms, reflecting the time constants of recovery from IM and IS, respectively. The fractions of channels recovering from IM and from IS were 0.36 ± 0.04 and 0.64 ± 0.04, respectively (A1 and A2 in eq. 1). Addition of 300 µM lidocaine to the bath had no significant effect on the time constants, and the relative amplitudes of IM and IS (1 = 793 ± 206 ms and 2 = 5542 ± 781 ms, A1 = 0.34 ± 0.06 and A2 = 0.66 ± 0.06). These data show that the relative distribution of channels between IM and IS remained unchanged by lidocaine. If lidocaine had a higher affinity to either one of the two states, then this state would be stabilized relative to the state with the lower affinity, resulting in a change of the relative distribution of channels between the two states. Since this is not the case, we conclude that the affinity of lidocaine to IM and IS must be similar. Thus IM and IS represent high-affinity binding states for lidocaine.
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A Model for Lidocaine Interaction with the Cytoplasmic Pore. In a previous study, we demonstrated that binding of the inactivation gate to the cytoplasmic vestibule of the voltage-gated Na+ channel can protect the channels from entry into IUS (Hilber et al., 2002). We suggested that IUS may reflect a dynamic rearrangement involving the cytoplasmic vestibule of the channel. When the inactivation gate is bound to the cytoplasmic vestibule, it may stabilize the structure of the cytoplasmic vestibule and thereby prevent channels from entering into IUS. The data presented in this report indicate that lidocaine binds to a site in the cytoplasmic vestibule of the channel and interferes with the pore motion that underlies IUS.
Which mechanism might underlie the interaction of lidocaine with IUS? We propose a simple mechanism in which we assume that the lidocaine-bound states and the IUS state are mutually exclusive.
This hypothetical mechanism can be represented by Scheme 1, where IM,S represents the high-affinity states for lidocaine binding (IM and IS), IM,S-lido represents the lidocaine-bound inactivated states, IUS is the ultra-slow inactivated state, and k represents the forward rate constant into the IUS. The transitions leading from the closed state to IM,S have been omitted because they are very rapid compared with the development of the IUS state. IUS is absorbing (Fig. 5A and see below), therefore the rate of exit from IUS can be assumed to be zero. This scheme predicts that the apparent time constant of entry into IUS should increase in a concentration-dependent manner. To test this prediction, we determined the time course of entry into IUS in the following manner: prepulses to –20 mV of variable duration were applied to K1237E channels from a holding potential of –120 mV, and the time course of recovery at –120 mV was monitored for each prepulse duration. The time course of recovery for each prepulse duration was then fitted with two exponentials that yielded time constants and amplitudes of IS and IUS. To derive the apparent time constant of entry into IUS (entry), the fraction of channels recovering from IUS following a given prepulse duration (A2) was plotted as a function of the respective prepulse duration. These data were well fitted with a monoexponential function (eq. 2) allowing for estimation of entry, which was 348 ± 29 s under control conditions and 649 ± 44 s during exposure to 10 µM lidocaine (Fig. 5A). Figure 5A also demonstrates that IUS is absorbing under drug-free conditions and during exposure to lidocaine. In Fig. 5B, the drug-induced change in entry is plotted as a function of the lidocaine concentration. Clearly, lidocaine produces a concentration-dependent increase in entry.
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If binding and unbinding of lidocaine is fast compared with the development of IUS, the above scheme also predicts that the apparent time constant of entry into IUS (entry) will be increased by exactly the same factor by which the fraction of channels recovering from IM,S is increased by lidocaine: -fold change in the fraction of blocked channels (IM,S-lido) = entry (during lidocaine exposure)/entry (under lidocaine free conditions).
This relationship allows the estimation of KD for lidocaine binding to the inactivated high-affinity states (IM,S). The KD for lidocaine binding, derived by this method, was 12.8 ± 2.5 µM (Fig. 5B). This value is in good agreement with the KD for occupancy of the IM state by lidocaine (Fig. 3C), which strongly supports the validity of the proposed model. (As shown in Fig. 4, the KD for lidocaine binding to IS is similar to the KD for lidocaine binding to IM).
Lidocaine Has Similar Effects on IUS in K1237E and K1237S. The amino acid Lys1237 is a part of the selectivity filter of the Na+ channel (Lipkind and Fozzard, 1994). Previously, Sunami et al. (1997) suggested that this residue electrostatically interacts with the binding of lidocaine. We wondered whether in the mutant K1237E an electrostatic interaction between lidocaine and Glu1237 plays a role in the modulation of IUS by lidocaine. Therefore, we examined the effect of lidocaine on IUS in a mutant in which the positively charged Lys1237 was replaced by the neutral amino acid serine. As shown in Fig. 6, the effect of 300 µM lidocaine on IUS in K1237S was similar to the effect of lidocaine on IUS in the mutant K1237E (Fig. 1C). The fact that the kinetic effect of lidocaine was not influenced by the charge of residue 1237 argues against the notion that the effect of lidocaine on IUS is the result of a direct interaction of lidocaine with the selectivity filter region.
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Discussion |
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) and thus allows for a more accurate quantitative analysis.
Local anesthetic drugs like lidocaine have long been known to produce voltage- and frequency-dependent block of Na+ channels (Weidmann, 1955). It has been proposed that local anesthetic drugs bind with high affinity to the fast inactivated conformational state, resulting in stabilization of this state. Upon repolarization the drug-bound fast inactivated state recovers more slowly than the drug-free fast inactivated state, giving rise to frequency-dependent block (Hille, 1977; Hondeghem and Katzung, 1977; Bean et al., 1983).
This view has recently been challenged by the observation that the time course of opening of the fast inactivation gate during repolarization is unchanged by lidocaine (Vedantham and Cannon, 1998). Alternatively, it has been suggested that local anesthetics bind to and stabilize slower inactivated states (IM, IS) (Fakler et al., 1990; Kambouris et al., 1998; Chen et al., 2000; Ong et al., 2000). In this case, recovery from inactivation is prolonged because lidocaine increases the number of channels entering these long lived inactivated states at depolarized potentials. The IM state is characterized by time constants of recovery on the order of 100 to 1000 ms (Kambouris et al., 1998). We show that binding of lidocaine to IM is preserved in the mutation K1237E (Fig. 3). Furthermore, as shown in Fig. 4, channels residing in the IS state seem to bind lidocaine with similar affinity as channels in the IM state, which is consistent with data obtained in wild-type NaV 1.4 channels (Ong et al., 2000). This result is not unexpected since lidocaine can only prolong the development of the IUS state if it is still bound to the channel during the time course of entry into this state. The IUS state takes several minutes to develop (Fig. 5A), thus most channels will reside in the IS state during the entry phase into IUS.
The interaction of lidocaine with the IUS state was mediated by binding of the drug to the residue Phe1579 in the inner vestibule of the channel as the double mutant K1237E + F1579A substantially reduced the modulation of IUS by lidocaine. Apart from binding to Phe1579, interactions of lidocaine with residues in S6 segments of domains I and III have also been reported (Wright et al., 1998; Yarov-Yarovoy et al., 2001, 2002). However, these interactions are substantially weaker than the interaction with Phe1579 (Ragsdale et al., 1996; Yarov-Yarovoy et al., 2002). Nevertheless, we cannot exclude that residues in other domains may play an additional role in the modulation of IUS by lidocaine.
Binding of lidocaine resulted in an increase in the apparent time constant of entry into IUS (Fig. 5), whereas the time course of recovery from IUS remained unchanged (Table 1). These results could be explained if the lidocaine-bound states and the IUS state were mutually exclusive. In this case, binding of lidocaine would reduce the number of channels available to enter into IUS, thereby prolonging entry into IUS, whereas recovery from IUS remains unchanged. Application of this model to our experimental data allowed for a correct prediction of the KD for interaction of lidocaine with the IM state, which serves as a successful consistency check of the model.
Our model assumes high-affinity binding of lidocaine to slow inactivated states. Alternatively, it may be hypothesized that lidocaine binds to and stabilizes the IF state. In this case, dissociation of lidocaine would be the rate-limiting step during recovery from inactivation. The concentration-dependent increase in the amplitude of a component recovering with a time constant of 1000 ms (Fig. 3C, Table 2) would then reflect dissociation of lidocaine from blocked fast inactivated channels. It may be argued that if fast inactivated channels were unable to enter the IUS state, then stabilizing channels in the fast inactivated state would reduce the number of channels in the IUS state by mass action. On the other hand, ultra-slow inactivation was produced by holding the channels at –20 mV for 300 s. At such a depolarized potential, all channels enter fast inactivation, even in the absence of lidocaine (see Fig. 1 in Todt et al., 1999). Nevertheless, 100% of K1237E channels were able to enter into ultra-slow inactivation if the duration of the conditioning prepulse was increased to 2000 s, irrespective of whether lidocaine was present or absent (Fig. 5A). Thus, lidocaine could not have increased the number of channels entering IF at –20 mV, and entry into IF could not have prevented channels from entry into IUS during long conditioning prepulses. As mentioned above, Vedantham and Cannon (1998) have shown that lidocaine-induced slowing of Na+ channel repriming does not result from a slowing of recovery of the fast inactivation gate, which argues against a direct interaction between lidocaine binding and IF. This notion is supported by a more recent study demonstrating that the action of lidocaine on gating charge cannot be interpreted as stabilizing the Na+ channel in an IF state (Sheets and Hanck, 2003). Accordingly, a number of studies have postulated a linkage between IS and use-dependent local anesthetic block (for review, see Ong et al., 2000). Therefore, we interpret our data by assuming that lidocaine binds with high affinity to slower inactivated states (IM, IS).
Which molecular mechanism may account for the failure of lidocaine-bound channels to enter into IUS? Slow inactivated states have been suggested to reflect a closure of the outer vestibule of the channel (Tomaselli et al., 1995; Balser et al., 1996; Kambouris et al., 1998; Todt et al., 1999; Ong et al., 2000; Hilber et al., 2001, 2002; Vilin et al., 2001; Xiong et al., 2003). Specifically, both the IM state and the IUS state are sensitive to mutations in the outer vestibule of the channel (Balser et al., 1996; Todt et al., 1999; Hilber et al., 2001), and the IUS state can be modified by binding of a mutated µ-conotoxin GIIIA to the outer vestibule (Todt et al., 1999; Hilber et al., 2001). Recently, Ong et al. (2000) tested the state-dependent accessibility of an engineered cysteine in the outer vestibule to sulfhydryl-modifying agents. It was found that slow inactivation and lidocaine block inhibited side chain accessibility, suggesting that lidocaine block and slow inactivation share a common outer pore structural rearrangement. These data suggest that IM and IUS may reflect conformational changes of the outer vestibule. Lidocaine may bind to the cytoplasmic side of the selectivity filter, thereby stabilizing the outer vestibule (as suggested in Ong et al., 2000), resulting in a reduction of the IUS state as observed in this study. However, as shown in Fig. 6, lidocaine exerted similar effects on IUS irrespective of the charge at position 1237 in the selectivity filter. This suggests that the modulation of IUS by lidocaine does not result from an interaction with the outer vestibule of the channel.
The notion that slow inactivation involves a closure of the outer vestibule has recently been challenged by the demonstration that accessibility of cysteines, engineered to some positions in the outer vestibule, to the charged sulfhydryl-modifying agent methanethiosulfonate ethyltrimethylammonium (MTS-ET) was preserved during slow inactivation (Struyk and Cannon, 2002); however, only residues external to the selectivity filter (DEKA motif, inner ring of charge) could be studied. Hence, if slow inactivation encompassed a closure of the outer vestibule, this closure most likely occurs at the level of the selectivity filter. In support of this notion, the mutation W402C, located close to the selectivity filter region, substantially reduced IM (Balser et al., 1996).
Entry into IUS is inhibited by binding of a mutated µ-conotoxin GIIIA to the outer vestibule (Todt et al., 1999; Hilber et al., 2001). µ-Conotoxin GIIIA does not interact with the selectivity filter (Dudley et al., 2000), which is inconsistent with the idea that IUS solely reflects a localized constriction of the selectivity filter. If IUS encompassed a motion of the outer vestibule as suggested by the modulatory action of µ-conotoxin GIIIA, but the accessibility of the outer vestibule remained intact during prolonged depolarization as suggested by the data of Struyk and Cannon (2002) then it follows that the motion underlying IUS might not be a closure of the outer vestibule. Alternatively, the outer vestibule might become wider during IUS, which is consistent with the demonstration that the outer vestibule is a very flexible structure (Benitah et al., 1997). But how can a widening of the outer vestibule produce a long-lived nonconducting state? Recent data from a crystallized voltage-gated K+ channel suggest that gating involves a complex rearrangement of the channel protein. Opening of K+ channels most likely results from a motion during which the S5 helices are pulled away from the pore axis, which causes the S6 helices to move apart resulting in opening of the pore (Jiang et al., 2003b). Hence motions of the S5 segments and, most likely, of the adjacent P-loops seem to be transmitted to the S6 segments.
From these findings, we speculate that P-loops exert a lateral motion during IUS associated with a widening of the outer pore, which is transmitted from the P-loops to the adjacent S6 segments, resulting in a closure of the inner vestibule of the channel (Fig. 7). Binding of lidocaine to the inner vestibule of the channel might interfere with this closure of the inner vestibule by a foot-in-the-door mechanism thereby preventing the channels from entry into IUS. Such a foot-in-the-door model would be consistent with the fact that lidocaine slowed entry into IUS but did not affect recovery from IUS and would explain why the lidocaine-bound inactivated states (IM,IS) and the IUS state are mutually exclusive. A similar model has previously been proposed for the interaction of tetraethylammonium with the fast inactivated state in Shaker K+ channels (Choi et al., 1991).
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The idea that IUS involves a rearrangement of the inner vestibule of the channel is supported by the following findings. First, an interaction of the inactivation particle with the inner vestibule modulates IUS in a mutant in which IUS is not absorbing (Hilber et al., 2002). Second, a number of mutations in S6 segments have been reported to affect slow inactivated states (Wang and Wang, 1997; Takahashi and Cannon, 1999; O'Reilly et al., 2001). Third, G protein-coupled receptor- and protein kinase-dependent reductions in Na+ channel availability have recently been shown to be mediated by modulation of slow inactivation (Carr et al., 2003), consistent with a significant role of the internal parts of the Na+ channel in slow inactivation. Fourth, in KV 1.4 channels, slow inactivation is associated with a decrease in intracellular aqueous pore volume (Jiang et al., 2003a), again pointing toward a significant role of the inner vestibule in slow inactivation gating. If the proposed model holds true then slow inactivated states may represent a heterogeneous group of molecular rearrangements, some involving the outer pore (IM) and others the inner vestibule (IUS). Given the importance of slow inactivation in a great number of disease states (Goldin, 2003), a detailed understanding of the mechanistic basis of slow inactivated states has significant implications with regard to designing pharmacologic modulators of slow inactivation.
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Acknowledgements |
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Footnotes |
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W.S. and J.S. contributed equally to this work.
ABBREVIATIONS: IUS, ultra-slow inactivation; IM, intermediate inactivation; IS, slow inactivation; IM,S, intermediate and slow inactivation.
Address correspondence to: Dr. Hannes Todt, Medical University of Vienna, Institute of Pharmacology, Währingerstrasse 13A, A-1090 Vienna, Austria. E-mail: hannes.todt{at}meduniwien.ac.at
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