Compound-specific Na+ channel pore conformational changes induced by local anaesthetics
Departments of Anaesthesiology Pharmacology, Vanderbilt University, School of Medicine, Nashville, TN 37232, USA
Abstract
Upon prolonged depolarizations, voltage-dependent Na+ channels open and subsequently inactivate, occupying fast and slow inactivated conformational states. Like C-type inactivation in K+ channels, slow inactivation is thought to be accompanied by rearrangement of the channel pore. Cysteine-labelling studies have shown that lidocaine, a local anaesthetic (LA) that elicits depolarization-dependent (‘use-dependent’) Na+ channel block, does not slow recovery from fast inactivation, but modulates the kinetics of slow inactivated states. While these observations suggest LA-induced stabilization of slow inactivation could be partly responsible for use dependence, a more stringent test would require that slow inactivation gating track the distinct use-dependent kinetic properties of diverse LA compounds, such as lidocaine and bupivacaine. For this purpose, we assayed the slow inactivation-dependent accessibility of cysteines engineered into domain III, P-segment (μ1: F1236C, K1237C) to sulfhydryl (MTSEA) modification using a high-speed solution exchange system. As expected, we found that bupivacaine, like lidocaine, protected cysteine residues from MTSEA modification in a depolarization-dependent manner. However, under pulse-train conditions where bupivacaine block of Na+ channels was extensive (due to ultra-slow recovery), but lidocaine block of Na+ channels was not, P-segment cysteines were protected from MTSEA modification. Here we show that conformational changes associated with slow inactivation track the vastly different rates of recovery from use-dependent block for bupivacaine and lidocaine. Our findings suggest that LA compounds may produce their kinetically distinct voltage-dependent behaviour by modulating slow inactivation gating to varying degrees.
, 百拇医药
Introduction
Local anaesthetics (LAs) inhibit voltage-dependent Na+ channels, and are widely used to provide regional anaesthesia, and treat disorders of cellular excitability (epilepsy, cardiac arrhythmias). Although LAs are clinically important, the detailed mechanisms by which these drugs exert their distinctive inhibitory effects on Na+ channels are still debated. Upon membrane depolarization, Na+ channels rapidly undergo voltage-dependent conformational changes that lead to channel activation. Simultaneously, depolarization triggers initiation of fast inactivation, a process that terminates Na+ ion influx (Hodgkin & Huxley, 1952), and involves structural changes in the III–IV linker region (West et al. 1992). A fundamental feature of LAs is their ‘use-dependent’ blocking property (Courtney, 1975) that elicits cumulative block during trains of depolarizing pulses. Earlier experiments that showed a hyperpolarizing shift of the steady-state Na+ channel availability curve (Hille, 1977; Bean et al. 1983) and LA resistance of Na+ channels defective in fast inactivation (Cahalan, 1978; Yeh & Tanguy, 1985; Wang et al. 1987) suggested that use dependence might arise from LA-induced stabilization of fast inactivation. However, recent studies showing accessibility of III–IV linker fast inactivation gating structures to sulfhydryl modification is insensitive to use-dependent LA block have challenged this hypothesis (Vedantham & Cannon, 1999).
, 百拇医药
With prolonged depolarizations, Na+ channels progressively enter ‘slow inactivated’ states with lifetimes ranging from hundreds of milliseconds to several seconds (Adelman & Palti, 1969). Pore mutations and ionic conditions that modify slow inactivation have parallel effects on Na+ channel sensitivity to use-dependent block by LAs (Kambouris et al. 1998; Chen et al. 2000). In Shaker K+ channels, conformational changes in the pore structure have been linked to slow ‘C-type’ inactivation (Choi et al. 1991; Hoshi et al. 1991; Lopez-Barneo et al. 1993; Yellen et al. 1994). In Na+ channels, linkages between the position of pore residues near the selectivity filter, DEKA (Heinemann et al. 1992), and slow inactivation have also been identified (Benitah et al. 1999; Todt et al. 1999; Ong et al. 2000; Hilber et al. 2001). A recent study suggests pore residues C-terminal to the selectivity filter are not involved in slow inactivation (Struyk & Cannon, 2002). Hence, it appears that residues in or N-terminal to the Na+ channel selectivity filter may represent the narrowest regions of the pore and are the most affected by slow inactivation gating.
, 百拇医药
Recently, we showed that slow inactivation protected an engineered cysteine at the 1236 position of the rat skeletal muscle Na+ channel (μ1: F1236C) from depolarization-dependent MTSEA modification (Ong et al. 2000). Lidocaine-induced use-dependent block substantially increased cysteinyl protection during pulse protocols optimized to elicit slow inactivation, but not fast inactivation. However, to clearly establish a causal link between slow inactivation and use-dependent block, it is necessary to show that the LA-associated protection of P-segment cysteines tracks the use-dependent properties of LA compounds with distinctive kinetic signatures such as lidocaine and bupivacaine.
, 百拇医药
Here we assayed the state-dependent accessibility of two cysteines engineered into the domain III, P-segment (μ1: F1236C, K1237C) to sulfhydryl modification with a methanethiosulphonate (MTS) reagent, MTS-ethylammonium (MTSEA). The rat skeletal muscle sodium channel (μ1) was used because this isoform has been well characterized especially with regard to slow inactivation and local anaesthetic block. Furthermore, previous studies have shown that these channels respond linearly to MTSEA reagents (Perez-Garcia et al. 1997). Using lidocaine and bupivacaine in a high-speed solution exchange system we are able to show, under identical pulse-train conditions, that sulfhydryl protection is conditionally dependent on the degree of use-dependent block. We propose that LA compounds may produce their kinetically distinct voltage-dependent behaviour by modulating slow inactivation gating to varying degrees.
, http://www.100md.com
Methods
Mutagenesis and heterologous expression
The rat skeletal muscle Na+ channel subunit (μ1) was subcloned into the HindIII–XbaI site of the vector green fluorescent protein with an internal ribosomal entry site (pGFPires) as previously described (Johns et al. 1997). Mutagenesis, F1236C or K1237C, was performed on the rat Na+ channel (Yamagishi et al. 1997) using a PCR-based method (QuickChange site-directed mutagenesis kit; Stratagene, La Jolla, CA, USA). All mutations were confirmed by dideoxynucleotide sequencing. Wild-type and mutated channels were expressed in HEK 293 cells using lipofectamine (Invitrogen Co., Carlsbad, CA, USA) along with Na+ channel 1 subunit (provided by Dr Alfred George, Vanderbilt University). For experiments with F1304Q (provided by Dr Gordon Tomaselli, Johns Hopkins University), cells were cotransfected with rat Na+ channel 1 subunit, and GFP. Transfected cells were incubated at 37°C in 95% O2–5% CO2 for 24–72 h before electrical recordings. Cells exhibiting green fluorescence were chosen for electrophysiological analysis.
, 百拇医药
Electrophysiology
All recordings were performed at room temperature (20–22°C). Whole-cell Na+ currents (INa) were obtained using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA) and were acquired using pCLAMP 8 (Axon Instruments). Patch pipettes were pulled from borosilicate glass (Warner Instrument Inc., Hamden, CT, USA) with a Model P-97 Flaming-Brown micropipette puller (Sutter Instruments, San Rafael, CA, USA). Pipette resistance was 1–3 M when filled with a pipette solution containing (mM): 140 NaF, 10 NaCl, 5 EGTA, 10 Hepes, pH 7.40. Replacing the intracellular K+ with Na+ eliminated the time-dependent K+ currents in our HEK cell recordings. For experiments involving F1304Q channels, pipette solution contained (mM): 10 NaF, 110 CsF, 20 CsCl, 10 EGTA, 10 Hepes, pH 7.35 with CsOH. The bath solution in both cases contained (mM): 150 NaCl, 4.5 KCl, 1.5 CaCl2, 1 MgCl2, 10 Hepes (pH 7.4 with NaOH). In all recordings, 80% of the series resistance was compensated, yielding a maximum voltage error of 1 mV. MTS reagents: MTSEA, MTS-ethylsulphonate (MTSES), and MTS-ethyltrimethylammonium (MTSET) (Toronto Research Chemicals, Toronto, Ontario, Canada) were kept on ice as high concentration stock solutions and were diluted to 200 μM, 300 μM, or 5 mM in 5 ml bath solution immediately before use. Lidocaine HCl (Sigma Chemical Co., St Louis, MO, USA) or bupivacaine HCl (Sigma Chemical Co.) were diluted from stock solutions to the bath concentrations indicated in the text. Cells were clamped in whole-cell mode for at least 10 min before recording data. Whole-cell currents were sampled at 20 kHz (DigiData 1200 A/D converter; Axon instruments) and low pass-filtered at 5 kHz.
, 百拇医药
Rapid solution exchange
Rapid solution exchange was achieved using a computer-controlled High Speed Solution Exchange system (HSSE) (ALA Scientific Instruments, Westbury, NY, USA). HSSE has two orthogonal microbore Teflon output tubes (300 μm inner diameter) which face each other at 90 deg in the same plane (as depicted in Fig. 1A). The pipette (along with the cell attached) was positioned close to the tips of the tubes. The solution flow through the two tubes was controlled using pCLAMP 8.0 software (Axon instruments). The time resolution of HSSE in our set-up was determined by exposing a high Na+ bath solution (150 mM NaCl) and a low Na+ bath solution (10 mM NaCl and 135 mM N-methyl-D-glucamine+) alternatively to wild-type Na+ channels using the protocol shown in Fig. 1B (inset). Briefly, a test pulse to –20 mV was applied in the presence of high Na+. After 50 ms at –120 mV the cell was exposed to the low Na+ solution for varying durations (P1). As seen in Fig. 1B, the direction of the Na+ current fully reversed by the time the cell was exposed to low Na+ for 30 ms, suggesting that solution exchange was completed within this time frame. The HSSE system was used only in experiments shown in Figs 4 and 5.
, 百拇医药
A, two orthogonal microbore tubes were positioned at right angles and in close proximity to the recording pipette and cell. Solution flow was switched between the tubes using signals from pCLAMP software. B, the minimum solution exchange time was determined using the protocol shown in the inset. Wild-type Na+ currents were measured using a double depolarization pulse protocol. The first pulse was applied in high Na+ solution, while the second pulse was applied in low Na+ solution after a variable P1 interval. Peak INa decreases and reverses direction within 30 ms after switching to low Na+ solution. C, voltage dependence of activation and inactivation of K1237C channels obtained using the protocols shown in the inset and fitted to a Boltzmann function as described in the text.
, http://www.100md.com
A, protocol used to examine the effects of use-dependent block on the MTSEA modification of K1237C. Peak INa was recorded during 100 repetitive pulses to –20 mV in the presence of lidocaine or bupivacaine and in the presence or absence of MTSEA. Repetitive pulses to –20 mV for 1 s were applied with a 3 s interpulse repolarization period (at –120 mV) to selectively allow recovery from lidocaine, but not bupivacaine block (see rectangle in Fig. 3A). B, in the absence of MTSEA, the data confirm nearly complete recovery from lidocaine block using a 3 s repolarization period (peak INa reduced only 10% even after 100 pulses), in contrast to bupivacaine (INa reduced 50% after 100 pulses). C, peak INa recorded in the presence of MTSEA using the protocol shown in panel A. Plotted are currents recorded before exposure to MTSEA (Pre) and 60 s after the pulse train (Post), overlaid for comparison. MTSEA was applied from 100 ms before the depolarization to 400 ms into the depolarization (a total of 500 ms) using the HSSE system, as indicated in the protocol shown in panel A. D, fractional modification by MTSEA is summarized in the bar graph as the percentage reduction of peak INa (1 – Post/Pre). The number of cells recorded in each condition is shown in parentheses. Enhanced slow inactivation in the presence of bupivacaine reduced modification of K1237C by MTSEA compared to lidocaine or control (P < 0.05).
, http://www.100md.com
A, development of LA block was evaluated using the protocol shown in the inset. Both 300 μM lidocaine () and 30 μM bupivacaine () produced similar levels of block of INa (shaded region) after prolonged depolarizations (500–1000 ms). B, peak INa recorded using the protocol shown in the panel. In both LAs 50 repetitive pulses were applied to –20 mV with a 3 s interpulse recovery at –120 mV. Here, MTSEA (5 mM) was applied for the latter 500 ms of each 1 s depolarization, using the HSSE system, to expose cells to MTSEA during similar LA block. Plotted are currents recorded before (Pre) and 60 s after pulse train (Post), overlaid for comparison. C, fractional modification by MTSEA is summarized in the bar graph as the percentage reduction of peak INa (1 – Post/Pre). The number of cells recorded in each condition is shown in parentheses. For similar levels of slow inactivation, bupivacaine and lidocaine protected K1237C residue from MTS modification to a similar extent (NS with each other but P < 0.05 with control).
, 百拇医药
Data analysis
The data were acquired and analysed using pCLAMP 8.0 software (Axon instruments). All results are expressed as means ± S.E.M. and statistical comparisons were made using one-way ANOVA (Origin, OriginLab Corp., Northampton, MA, USA) with P < 0.05 indicating significance. Multi-exponential functions were fitted to the data using nonlinear least-squares methods (Origin).
Results
We expressed rat skeletal muscle Na+ channel (μ1) in HEK-293 cells to allow whole-cell voltage-clamp measurements. We measured peak INa at varying activating voltages and after conditioning steps to a range of inactivating voltages (protocol inset, Fig. 1C). The voltage dependence of activation (open squares) and inactivation (filled squares) were plotted, and voltage-dependent parameters were derived by fitting a Boltzmann function:
, 百拇医药
where V1/1 is the half-maximal voltage and k is the slope factor. The V1/1 of activation was –31.6 ± 1.3 (n = 12) while the V1/1 of inactivation was –74 ± 0.7 (n = 10), consistent with previously published values (Chahine et al. 1996). Cummins et al. (1999) reported that HEK-293 cells have endogenous Na+ current with V1/1 of activation of –42.4 ± 3.2 mV and V1/1 of inactivation of –88.4 ± 1.9 mV. However, the values obtained from our experiments (see above) suggest that the current was predominantly due to the expressed rat skeletal muscle Na+ channels and not the endogenous channels.
, 百拇医药
Depolarization-dependent accessibility of pore cysteines to MTS reagents
To assess conformational changes in the pore, we first examined depolarization-dependent accessibility of pore cysteine mutations to sulfhydryl reagents. For comparison to our prior studies (Ong et al. 2000), we retested the accessibility of a cysteine engineered at the 1236 locus (F1236C), to sulfhydryl reagents using the same protocols from Ong et al. (Fig. 2A). Since prior studies (Perez-Garcia et al. 1997) showed that MTSEA modification reduced peak INa in a voltage-independent manner, all of our experiments were conducted with depolarizations to –20 mV, a potential at which peak inward current was maximum (data not shown). A 3 min exposure to 200 μM MTSEA during prolonged depolarization (protocol II) reduced F1236C peak sodium current (31 ± 3%) to a greater extent than hyperpolarization (protocol I) (20 ± 1%, P < 0.05). While these results reconfirm that depolarization increases the accessibility of the cysteine side chain (see Fig. 2 of Ong et al. 2000), they do not distinguish between changes in MTS reactivity due to fast or slow inactivation. The higher concentration of MTSEA required for depolarization-dependent modification of F1236C in this study was probably due to variation in the potency of the compound.
, 百拇医药
A, peak INa was recorded from HEK-293 cells transfected with F1236C or K1237C mutant channels during a control period, or in lidocaine (100 μM) or bupivacaine (10 μM). State-dependent modification of these cysteines by MTSEA was assessed with the two protocols (I and II) shown. After an initial pulse (P1) to –20 mV, MTSEA was washed into the bath. After a 1 min equilibration period, the membrane was hyperpolarized to –120 mV (protocol I) or depolarized to –20 mV (protocol II) for 3 min. After MTSEA was washed out (1 min), INa was reassessed using a brief pulse to –20 mV (P2). B, representative currents through K1237C channels elicited by P1 and P2 pulses are superimposed for comparison. The left and middle traces were obtained using protocol I and II, respectively. The trace on the right was obtained using protocol II in the presence of lidocaine. C, summary data showing the fractional reduction of peak INa due to sulfhydryl modification (1 – P2/P1). MTSEA was applied to the F1236C (200 μM) or K1237C (300 μM) mutants, respectively. Depolarization facilitated MTSEA modification of K1237C (P < 0.0001) and F1236C (P < 0.05), while MTSET or MTSES modification of K1237C was minimal with depolarization. Lidocaine and bupivacaine reduced the MTSEA modification of both F1236C and K1237C during depolarization (P < 0.05).
, http://www.100md.com
We next examined the accessibility of a cysteine at the adjacent 1237 position. Since K1237 is located in the deepest region of Na+ channel domain III P-loop, and forms part of the ion selectivity filter (DEKA) (Heinemann et al. 1992; Tsushima et al. 1997), we hypothesized that a cysteine in this position might be less accessible in the baseline (hyperpolarized) state, while maintaining a similar degree of accessibility as F1236C in the depolarized state. As seen in Fig. 2B, and summarized in the bar graph in Fig. 2C, exposure of MTSEA during depolarization reduced K1237C peak INa more than during hyperpolarization (31 ± 2% versus 15 ± 2%, P < 0.0001), and the difference was slightly greater than at the 1236 position (Fig. 2C). While the extent of MTSEA modification is consistent with our earlier studies (Ong et al. 2000), the gating-dependent differences in accessibility are consistently modest. This suggests that slow inactivation is associated with subtle, yet distinguishable, conformational changes in the pore. Identical experiments using the larger, positively charged reagent MTSET and the negatively charged analogue MTSES were performed, but neither reagent produced depolarization-dependent modification of INa (Fig. 2C). The insensitivity of K1237C to these MTS reagents (MTSET and MTSES) is consistent with a previous study (Chiamvimonvat et al. 1996).
, http://www.100md.com
As shown previously for the 1236 locus (Ong et al. 2000), lidocaine exposure attenuated the modification of K1237C by MTSEA during depolarization (Fig. 2B and C, 20 ± 3%, versus control depolarization, P < 0.05). For consistency with prior work, the effect of lidocaine to attenuate MTSEA modification of the 1236 locus was also reconfirmed (Fig. 2C). In addition, bupivacaine, a LA with far slower use-dependent recovery kinetics, also attenuated MTSEA modification of K1237C (Fig. 2C, 19 ± 2%, versus control depolarization, P < 0.05). These results indicate that inhibition of the depolarization-dependent MTSEA modification is not specific to lidocaine, but also holds true for other LA compounds.
, 百拇医药
Kinetic differences between local anaesthestics
To explore whether kinetic differences between LA compounds in use-dependent block correlate with cysteine side chain accessibility in the P-loop during gating, we first evaluated the rate at which K1237C channels recovered from block by either lidocaine or bupivacaine (Fig. 3A). The recovery kinetics were fitted to a biexponential function (Table 1), and the drug concentrations were selected to produce slow recovery components of similar magnitude (A2). Lidocaine (100 μM) and bupivacaine (30 μM) increased the amplitude of the slow recovery component to a similar degree (A2: 0.78 ± 0.01 for lidocaine, 0.79 ± 0.04 for bupivacaine). However, the time constant of recovery of the slow component (slow) in bupivacaine was much slower than in lidocaine (Fig. 3A: slow; 8744 ± 444 ms versus 314 ± 14 ms, P < 0.0001). The slower recovery from bupivacaine block is in agreement with previous findings (Clarkson & Hondeghem, 1985; Berman & Lipka, 1994).
, 百拇医药
A, recovery from inactivation and drug block in K1237C channels evaluated using the protocol shown in the inset of panel B. Continuous lines are two exponential fits to the mean data: y = y0 + A1 (1 – e–t/fast) + A2 (1 – e–t/slow). Recovery from inactivation was significantly delayed in 30 μM bupivacaine () compared to control () or 100 μM lidocaine (). Within 3 s, Na+ channels completely recovered from lidocaine block, while only 50% of the channels recovered from bupivacaine block. B, recovery from inactivation and drug block of fast inactivation deficient mutant channels, F1304Q. Inset shows INa recorded from F1304Q and K1237C channels. Note the removal of fast inactivation in F1304Q channels. As in K1237C, recovery from inactivation in F1304Q channels was significantly delayed in 30 μM bupivacaine () compared to control () or 100 μM lidocaine (). All parameters are defined and summarized in Table 1.
, http://www.100md.com
To determine if the distinctive slow recovery components induced by lidocaine and bupivacaine require intact fast inactivation gating structures, we utilized a previously described mutant Na+ channel lacking fast inactivation, F1304Q (Balser et al. 1996b). Figure 3B inset shows normalized INa recorded from K1237C and F1304Q channels during a step depolarization to –20 mV from a holding potential of –120 mV. While K1237C channels completely inactivate within a few milliseconds, F1304Q channels do not inactivate even after 10 ms, demonstrating that F1304Q channels are fast inactivation deficient. We examined the recovery from lidocaine and bupivacaine block of F1304Q (Fig. 3B). As expected, the magnitude of the fast recovery component (A1) is markedly reduced in drug-free conditions (Table 1). Nonetheless, in bupivacaine, the time constant of recovery of the slow component (slow) remains long, and far slower than in lidocaine (Fig. 3B: slow; 5088.5 ± 442.5 ms versus 167 ± 30.3 ms, P < 0.01). Moreover, recovery of the F1304Q slow component in lidocaine was still delayed compared to drug-free conditions (see Table 1). These results show that distinctive recovery components induced by these LA agents do not require intact fast inactivation, and corroborate the findings of Vedantham & Cannon (1999).
, 百拇医药
Slow inactivation protects MTS modification
Figure 3A also shows that 3 s after the end of the 1 s conditioning pulse, almost all K1237C channels had fully recovered (–120 mV) in the presence of lidocaine, while only 40–50% of channels recovered in the presence of bupivacaine (vertical bar, Fig. 3A). We made use of this difference to identify pulse train conditions that render cumulative block in bupivacaine, but not lidocaine. The protocol (Fig. 4A) applied repetitive 1 s depolarizing pulses to –20 mV with an interpulse repolarization to –120 mV for 3 s, allowing nearly complete recovery between pulses in lidocaine, but only partial recovery in bupivacaine. Figure 4B shows the representative current (every 10th pulse) in the presence of lidocaine or bupivacaine, in the absence of MTSEA. Peak INa is reduced by 50% at steady state in the presence of bupivacaine (right), but only by 10% in the presence of lidocaine (left), consistent with the distinctive use-dependent characteristics of the two agents, as also reflected in Fig. 3A.
, http://www.100md.com
We then examined whether marked differences in cumulative block by bupivacaine and lidocaine predictably track inactivation-dependent sulfhydryl modification of the 1237 cysteinyl. To selectively examine accessibility of the 1237 cysteinyl during inactivation, a rapid solution exchange system was utilized (Fig. 1). MTSEA was applied from 100 ms before the depolarization to 400 ms after depolarization (a total of 500 ms), as indicated in the protocol shown in Fig. 4A. While these exposure periods include both fast and slow inactivation gating components, our results in F1304Q (Fig. 3B) effectively exclude fast inactivation as the gating component primarily responsible for lidocaine–bupivacaine kinetic differences. In these experiments, the concentration of MTSEA was increased to 5 mM to allow substantial modification during these brief exposure periods. Since MTSEA was applied only during the period shown using the HSSE system, non-specific effects were minimized. Although depolarization in control, lidocaine, or bupivacine would lead to slow inactivation of Na+ channels during the initial 400 ms of each depolarization, we hypothesized that cumulative use-dependent bupivacaine block (under the conditions of the protocol in Fig. 4A) would ‘protect’ the K1237C residue from modification. In contrast, the complete recovery from inactivation in drug-free conditions or in lidocaine would allow MTS modification of unblocked channels at the onset of each depolarization. The measured currents, recorded before (Pre) and 60 s after the 100 depolarizing pulses (Post) allow for complete recovery from use-dependent drug block, so that any current reduction reflects MTS modification (superimposed in Fig. 4C). The data from all experiments are summarized in the bar graph (Fig. 4D), which shows the percentage of channels that have been modified by MTSEA. We measured less modification of K1237C by MTSEA in bupivacaine (39 ± 4%) than in lidocaine or control (52 ± 3% and 57 ± 3%, respectively, both P < 0.05 versus bupivacaine). This result suggests that accessibility of the 1237 cysteine to MTSEA is more restricted under kinetic conditions where use-dependent bupivacaine block is maintained, but use-dependent lidocaine block is not.
, 百拇医药
A gating-independent explanation for this result is that bupivacaine somehow provides better protection of the cysteinyl side chain, independent of its effect on inactivation gating. In this case, we would anticipate greater protection from MTSEA modification by bupivacaine under conditions where use-dependent INa reduction is similar to that evoked by lidocaine. Figure 5A shows the development of use-dependent block, in the absence or presence of lidocaine or bupivacaine, evaluated using the protocol shown in the inset. The concentrations of lidocaine (300 μM) and bupivacaine (30 μM) were chosen so that most of the channels (> 85%) were blocked by 500 ms or longer depolarizations to –20 mV (boxed region in Fig. 5A). To evaluate cysteine modification, a repetitive 1 s depolarizing pulse was applied, and MTSEA was selectively exposed for 500 ms in the latter portion of the 1 s depolarization pulse (Fig. 5B, upper panel), a period when most of the Na+ channels were blocked by lidocaine or bupivacaine (boxed region, Fig. 5A). As in Fig. 4, a 3 s interpulse repolarization to –120 mV was applied between each depolarization. The optimal approach would allow 1 min or more for full recovery of all channels from bupivacaine block (Fig. 3A); however, given the lengthy duration of the experiment, this was technically not feasible. While a 3 s interpulse period allowed only 40–50% of channels to recover from bupivacaine block (versus 100% in lidocaine), during all subsequent depolarizations, the channels are nonetheless fully blocked by either LA during the critical period (the latter 500 ms of Pn) when MTSEA is repeatedly exposed. Hence, the protocol (Fig. 5B) does provide conditions where MTSEA is exposed to channels when the extent of slow inactivation is saturated for lidocaine and bupivacaine, and thus similar, particularly in contrast to the conditions of Fig. 4.
, 百拇医药
The modification by MTSEA (Fig. 5B, bottom panel) was assessed before (Pre) and 60 s after the pulse train (Post). As before, this allowed for complete recovery from use-dependent lidocaine and bupivacaine block, so that any current reduction reflects MTS modification alone. Lidocaine significantly inhibited the modification of MTSEA compared to control (Fig. 5C: 17 ± 3% versus 27 ± 3%, P < 0.05), and the reduction of MTSEA modification by lidocaine was comparable to that of bupivacaine (14 ± 3%, P < 0.05 with control, NS versus lidocaine). Hence, similar degrees of use-dependent block have comparable effects on cysteinyl modification. The findings suggest that the differences found in Fig. 4D relate to drug-induced differences in gating (differential recovery from slow inactivation), as opposed to direct LA-specific interference of cysteine modification.
, http://www.100md.com
Discussion
Pore rearrangement during slow inactivation
Voltage-gated Na+ channels are dynamic molecules that modify their conformational state in response to changes in membrane potential. Depolarization leads to channel inactivation within a few milliseconds (fast inactivation), a process involving residues situated near the cytoplasmic face of the channel (West et al. 1992). Prolonged depolarization induces channels to progressively occupy more stable slow-inactivated states. Numerous studies have linked conformational changes in the pore-lining segments of Na+ channels to slow inactivation (Balser et al. 1996a; Kambouris et al. 1998; Mitrovic et al. 2000; Xiong et al. 2003). The same residues, particularly K1237C, play a critical role in ionic selectivity of the pore for Na+ over Ca2+ (Favre et al. 1996; Tsushima et al. 1997).
, 百拇医药
Here we show that slow inactivation alters the accessibility of a cysteine engineered at the 1237 locus to sulfhydryl modification (Fig. 2), supporting prior studies indicating the deepest region of Na+ channel pore has restrictive access and undergoes conformational rearrangement during slow inactivation (Benitah et al. 1999; Ong et al. 2000). Unlike the K1237E or K1237S (Todt et al. 1999), K1237C in the present experiment did not produce ‘ultra slow’ inactivation ( on the order of 100 s), but rather, exhibited normal recovery kinetics in the absence of MTSEA (Fig. 3A), suggesting cysteine substitution alone did not significantly alter the channel gating. A recent study (Struyk & Cannon, 2002) showed that MTSET accessibility to pore cysteine mutations C-terminal to the selectivity filter did not change during slow inactivation. In our studies, MTSET (or MTSES) accessibility to K1237C was also not affected during depolarization (Fig. 2C) consistent with a previous study (Chiamvimonvat et al. 1996). Hence, pore rearrangements during slow gating appear to be focused on the narrow selectivity filter locus (K1237C) and residues immediately N-terminal (F1236C) (Ong et al. 2000) that is not accessible to the larger MTSET or negatively charged MTSES.
, http://www.100md.com
Local anaesthetics and slow inactivation
LAs exert their therapeutic effects by suppressing the ionic currents through Na+ channels. While the efficacy of LA action is enhanced by membrane depolarization (use dependence), the mechanistic basis for compound-specific differences in use-dependent drug block remains debated. Enzymes and mutations that remove fast inactivation attenuate use-dependent suppression of sodium channels by lidocaine and other Na+ channel blockers (Cahalan, 1978; Yeh & Tanguy, 1985; Bennett et al. 1995), suggesting an interaction between LAs and the fast-inactivated state. However, recent studies have questioned the postulated linkage between fast inactivation and use-dependent block by demonstrating that structural components of the Na+ channel involved in fast inactivation gating are not trapped in the fast-inactivated state by lidocaine (Vedantham & Cannon, 1999). In the present work, removal of fast inactivation (using a fast inactivation deficient mutant) did not eliminate the slow component of recovery seen in lidocaine and bupivacaine. Moreover, lidocaine did not inhibit the covalent modification of 1237C when MTSEA was exposed during the initial 400 ms of depolarization, a period that includes fast inactivation (Fig. 4), further supporting the findings of Vedantham & Cannon (1999). At the same time, bupivacaine elicits a slowly recovering block component within this time frame (Fig. 3A), and protected the 1237 cysteine from MTSEA modification (Fig. 4C and D).
, http://www.100md.com
Using MTSEA modification of the adjacent cysteine (F1236C), we recently (Ong et al. 2000) showed that lidocaine attenuates MTSEA modification during sustained depolarizations, but not brief, 5 ms depolarizations, also suggesting that pore rearrangements linked to slow inactivation and use-dependent lidocaine action are mechanistically linked (Ong et al. 2000). In this study, we provide further insight into the relationship between the pore rearrangement and use-dependent LA action by leveraging the kinetic differences between lidocaine and bupivacaine. Specifically, these data suggest that pore conformational changes that alter MTSEA accessibility track the differences in rate of recovery from use-dependent block for the two LAs and implicate slow inactivation in the compound-specific differences in use-dependent block.
, http://www.100md.com
We postulate the conceptual model shown in Fig. 6. While K1237C is relatively inaccessible during hyperpolarization (Fig. 6, left), depolarization leads to conformational changes in the pore-lining segments that increase MTSEA accessibility (Fig. 2). LAs do not inhibit accessibility of MTSEA during opening or fast inactivation, presumably because LAs are not trapped in the pore (Fig. 6, middle) (Vedantham & Cannon, 1999). However, with the accumulation of use-dependent drug block, the pore rearranges such that LA binding is stabilized and MTSEA accessibility of K1237C is reduced. Recently Bai et al. (2003) reported that mutations in the S6-domain LA binding sites affect slow inactivation, strengthening the notion that the binding of LAs may stabilize a structural rearrangement related to slow inactivation. Moreover, our findings showing the recovery kinetics of bupivacaine and lidocaine track the MTSEA accessibility of the 1237 cysteine side chain (Fig. 4) suggest that the various LAs may stabilize the slow-inactivated conformational state to differing degrees. As such, measured differences in rate of recovery from use-dependent block may reflect differences in LA affinity for the rearranged pore (Fig. 6, right).
, 百拇医药
The 1237C side chain is relatively inaccessible in hyperpolarized channels (left). Depolarization leads to conformational changes that increase accessibility of the cysteine residue (K1237C) to MTSEA modification, during channel opening and fast inactivation (middle). Prolonged depolarization leads to slow inactivation, which is accompanied by pore rearrangement. LAs stabilize pore rearrangement during slow inactivation (right), resulting in the reduction in the rate of MTSEA modification (ka > kb). We postulate that the extent of stabilization is dependent on the distinctive binding kinetics of the two LAs, as the dissociation rate of bupivacaine from its binding site is likely to be slower than that of lidocaine (i.e. Kd,bupi < Kd,lid).
, 百拇医药
Sunami et al. (1997) have shown electrostatic interactions between lidocaine binding and K1237, suggesting that the native lysine lies in close proximity to the LA-binding site in domain IV, segment 6 (Ragsdale et al. 1994). At the same time, the 1236 and 1237 cysteine side chains are accessible to only extracellular application of MTSEA (Yamagishi et al. 1997), while the LAs block the skeletal muscle isoform (used here) only from inside the cell (Sunami et al. 2000), indicating that a physical interaction whereby the LAs directly inhibit MTSEA activity across the selectivity filter is unlikely. Studies have shown that diverse drugs, including LAs, bind to the same residues in the transmembrane IV S6 segment of voltage-gated Na+ channels (Ragsdale et al. 1996). Mutation of these binding residues have distinct effects on different drugs, suggesting that the compounds may interact with the S6 domain in an overlapping but non-identical manner, which could depend on their chemical structure. It has also been postulated that LAs interact with hydrophobic aromatic residues (phenylalanine or tyrosine) through hydrophobic or electron interactions (Ragsdale et al. 1994). In this regard, bupivacaine has two aromatic rings while lidocaine has only one. Moreover, three hydrophobic aromatic residues face the pore lumen in the putative S6 LA binding site (Ragsdale et al. 1994). Since bupivacaine has two aromatic rings, it is possible that electron interactions are enhanced, and stabilize slow inactivation, thus delaying recovery from drug block. We postulate that LAs stabilize structural rearrangements coupled to slow inactivation by interaction with pore-lining side chains in or near the selectivity filter, and that use-dependent kinetic differences between LAs may partly result from differences in the stability of this drug-bound conformational state.
, http://www.100md.com
References
Adelman WJ Jr & Palti Y (1969). The effects of external potassium and long duration voltage conditioning on the amplitude of sodium currents in the giant axon of the squid, Loligo pealei. J General Physiol 54, 589–606.
Bai CX, Glaaser IW, Sawanobori T & Sunami A (2003). Involvement of local anesthetic binding sites on IVS6 of sodium channels in fast and slow inactivation. Neurosci Lett 337, 41–45.
, 百拇医药
Balser JR, Nuss HB, Chiamvimonvat N, Perez-Garcia MT, Marban E & Tomaselli GF (1996a). External pore residue mediates slow inactivation in mu1 rat skeletal muscle sodium channels. J Physiol 494, 431–442.
Balser JR, Nuss HB, Orias DW, Johns DC, Marban E, Tomaselli GF & Lawrence JH (1996b). Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels. J Clin Invest 98, 2874–2886.
, 百拇医药
Bean BP, Cohen CJ & Tsien RW (1983). Lidocaine block of cardiac sodium channels. J General Physiol 81, 613–642.
Benitah JP, Chen Z, Balser JR, Tomaselli GF & Marban E (1999). Molecular dynamics of the sodium channel pore vary with gating: interactions between P-segment motions and inactivation. J Neurosci 19, 1577–1585.
Bennett PB, Valenzuela C, Chen LQ & Kallen RG (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. Circ Res 77, 584–592.
, 百拇医药
Berman MF & Lipka LJ (1994). Relative sodium current block by bupivacaine and lidocaine in neonatal rat myocytes. Anesth Analg 79, 350–356.
Cahalan MD (1978). Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons. Biophys J 23, 285–311.
Chahine M, Deschene I, Chen LQ & Kallen RG (1996). Electrophysiological characteristics of cloned skeletal and cardiac muscle sodium channels. Am J Physiol 271, H498–H506.
, 百拇医药
Chen Z, Ong BH, Kambouris NG, Marban E, Tomaselli GF & Balser JR (2000). Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels. J Physiol 524, 37–49.
Chiamvimonvat N, Perez-Garcia MT, Ranjan R, Marban E & Tomaselli GF (1996). Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis. Neuron 16, 1037–1047.
Choi KL, Aldrich RW & Yellen G (1991). Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc Natl Acad Sci U S A 88, 5092–5095.
, http://www.100md.com
Clarkson CW & Hondeghem LM (1985). Mechanism for bupivacaine depression of cardiac conduction: fast block of sodium channels during the action potential with slow recovery from block during diastole. Anesthesiology 62, 396–405.
Courtney KR (1975). Mechanism of frequency-dependent inhibition of sodium currents in frog myelinated nerve by the lidocaine derivative GEA. J Pharmacol Exp Ther 195, 225–236.
Cummins TR, Dib-Hajj SD, Black JA, Akopian AN, Wood JN & Waxman SG (1999). A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons. J Neurosci 19, RC43.
, 百拇医药
Favre I, Moczydlowski E & Schild L (1996). On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel. Biophys J 71, 3110–3125.
Heinemann SH, Terlau H, Stuhmer W, Imoto K & Numa S (1992). Calcium channel characteristics conferred on the sodium channel by single mutations. Nature 356, 441–443.
Hilber K, Sandtner W, Kudlacek O, Glaaser IW, Weisz E, Kyle JW, French RJ, Fozzard HA, Dudley SC & Todt H (2001). The selectivity filter of the voltage-gated sodium channel is involved in channel activation. J Biol Chem 276, 27831–27839.
, 百拇医药
Hille B (1977). Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J General Physiol 69, 497–515.
Hodgkin AL & Huxley AF (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117, 500–544.
Hoshi T, Zagotta WN & Aldrich RW (1991). Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. Neuron 7, 547–556.
, 百拇医药
Johns DC, Nuss HB & Marban E (1997). Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs. J Biol Chem 272, 31598–31603.
Kambouris NG, Hastings LA, Stepanovic S, Marban E, Tomaselli GF & Balser JR (1998). Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat micro1 skeletal muscle sodium channel. J Physiol 512, 693–705.
, 百拇医药
Lopez-Barneo J, Hoshi T, Heinemann SH & Aldrich RW (1993). Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1, 61–71.
Mitrovic N, George AL Jr & Horn R (2000). Role of domain 4 in sodium channel slow inactivation. J General Physiol 115, 707–718.
Ong BH, Tomaselli GF & Balser JR (2000). A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence. J General Physiol 116, 653–662.
, http://www.100md.com
Perez-Garcia MT, Chiamvimonvat N, Ranjan R, Balser JR, Tomaselli GF & Marban E (1997). Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification. Biophys J 72, 989–996.
Ragsdale DS, McPhee JC, Scheuer T & Catterall WA (1994). Molecular determinants of state-dependent block of Na+ channels by local anesthetics. Science 265, 1724–1728.
Ragsdale DS, McPhee JC, Scheuer T & Catterall WA (1996). Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels. Proc Natl Acad Sci U S A 93, 9270–9275.
, http://www.100md.com
Struyk AF & Cannon SC (2002). Slow inactivation does not block the aqueous accessibility to the outer pore of voltagegated Na channels. J General Physiol 120, 509–516.
Sunami A, Dudley SC Jr & Fozzard HA (1997). Sodium channel selectivity filter regulates antiarrhythmic drug binding. Proc Natl Acad Sci U S A 94, 14126–14131.
Sunami A, Glaaser IW & Fozzard HA (2000). A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel. Proc Natl Acad Sci U S A 97, 2326–2331.
, 百拇医药
Todt H, Dudley SC Jr, Kyle JW, French RJ & Fozzard HA (1999). Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule. Biophys J 76, 1335–1345.
Tsushima RG, Li RA & Backx PH (1997). Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore. J General Physiol 109, 463–475.
Vedantham V & Cannon SC (1999). The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels. J General Physiol 113, 7–16.
, http://www.100md.com
Wang GK, Brodwick MS, Eaton DC & Strichartz GR (1987). Inhibition of sodium currents by local anesthetics in chloramine-T-treated squid axons. The role of channel activation. J General Physiol 89, 645–667.
West JW, Patton DE, Scheuer T, Wang Y, Goldin AL & Catterall WA (1992). A cluster of hydrophobic amino acid residues required for fast Na+-channel inactivation. Proc Natl Acad Sci U S A 89, 10910–10914.
Xiong W, Li RA, Tian Y & Tomaselli GF (2003). Molecular motions of the outer ring of charge of the sodium channel: do they couple to slow inactivation J General Physiol 122, 323–332.
, 百拇医药
Yamagishi T, Janecki M, Marban E & Tomaselli GF (1997). Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis. Biophys J 73, 195–204.
Yeh JZ & Tanguy J (1985). Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons. Biophys J 47, 685–694.
Yellen G, Sodickson D, Chen TY & Jurman ME (1994). An engineered cysteine in the external mouth of a K+ channel allows inactivation to be modulated by metal binding. Biophys J 66, 1068–1075., http://www.100md.com(Koji Fukuda, Tadashi Naka)
Abstract
Upon prolonged depolarizations, voltage-dependent Na+ channels open and subsequently inactivate, occupying fast and slow inactivated conformational states. Like C-type inactivation in K+ channels, slow inactivation is thought to be accompanied by rearrangement of the channel pore. Cysteine-labelling studies have shown that lidocaine, a local anaesthetic (LA) that elicits depolarization-dependent (‘use-dependent’) Na+ channel block, does not slow recovery from fast inactivation, but modulates the kinetics of slow inactivated states. While these observations suggest LA-induced stabilization of slow inactivation could be partly responsible for use dependence, a more stringent test would require that slow inactivation gating track the distinct use-dependent kinetic properties of diverse LA compounds, such as lidocaine and bupivacaine. For this purpose, we assayed the slow inactivation-dependent accessibility of cysteines engineered into domain III, P-segment (μ1: F1236C, K1237C) to sulfhydryl (MTSEA) modification using a high-speed solution exchange system. As expected, we found that bupivacaine, like lidocaine, protected cysteine residues from MTSEA modification in a depolarization-dependent manner. However, under pulse-train conditions where bupivacaine block of Na+ channels was extensive (due to ultra-slow recovery), but lidocaine block of Na+ channels was not, P-segment cysteines were protected from MTSEA modification. Here we show that conformational changes associated with slow inactivation track the vastly different rates of recovery from use-dependent block for bupivacaine and lidocaine. Our findings suggest that LA compounds may produce their kinetically distinct voltage-dependent behaviour by modulating slow inactivation gating to varying degrees.
, 百拇医药
Introduction
Local anaesthetics (LAs) inhibit voltage-dependent Na+ channels, and are widely used to provide regional anaesthesia, and treat disorders of cellular excitability (epilepsy, cardiac arrhythmias). Although LAs are clinically important, the detailed mechanisms by which these drugs exert their distinctive inhibitory effects on Na+ channels are still debated. Upon membrane depolarization, Na+ channels rapidly undergo voltage-dependent conformational changes that lead to channel activation. Simultaneously, depolarization triggers initiation of fast inactivation, a process that terminates Na+ ion influx (Hodgkin & Huxley, 1952), and involves structural changes in the III–IV linker region (West et al. 1992). A fundamental feature of LAs is their ‘use-dependent’ blocking property (Courtney, 1975) that elicits cumulative block during trains of depolarizing pulses. Earlier experiments that showed a hyperpolarizing shift of the steady-state Na+ channel availability curve (Hille, 1977; Bean et al. 1983) and LA resistance of Na+ channels defective in fast inactivation (Cahalan, 1978; Yeh & Tanguy, 1985; Wang et al. 1987) suggested that use dependence might arise from LA-induced stabilization of fast inactivation. However, recent studies showing accessibility of III–IV linker fast inactivation gating structures to sulfhydryl modification is insensitive to use-dependent LA block have challenged this hypothesis (Vedantham & Cannon, 1999).
, 百拇医药
With prolonged depolarizations, Na+ channels progressively enter ‘slow inactivated’ states with lifetimes ranging from hundreds of milliseconds to several seconds (Adelman & Palti, 1969). Pore mutations and ionic conditions that modify slow inactivation have parallel effects on Na+ channel sensitivity to use-dependent block by LAs (Kambouris et al. 1998; Chen et al. 2000). In Shaker K+ channels, conformational changes in the pore structure have been linked to slow ‘C-type’ inactivation (Choi et al. 1991; Hoshi et al. 1991; Lopez-Barneo et al. 1993; Yellen et al. 1994). In Na+ channels, linkages between the position of pore residues near the selectivity filter, DEKA (Heinemann et al. 1992), and slow inactivation have also been identified (Benitah et al. 1999; Todt et al. 1999; Ong et al. 2000; Hilber et al. 2001). A recent study suggests pore residues C-terminal to the selectivity filter are not involved in slow inactivation (Struyk & Cannon, 2002). Hence, it appears that residues in or N-terminal to the Na+ channel selectivity filter may represent the narrowest regions of the pore and are the most affected by slow inactivation gating.
, 百拇医药
Recently, we showed that slow inactivation protected an engineered cysteine at the 1236 position of the rat skeletal muscle Na+ channel (μ1: F1236C) from depolarization-dependent MTSEA modification (Ong et al. 2000). Lidocaine-induced use-dependent block substantially increased cysteinyl protection during pulse protocols optimized to elicit slow inactivation, but not fast inactivation. However, to clearly establish a causal link between slow inactivation and use-dependent block, it is necessary to show that the LA-associated protection of P-segment cysteines tracks the use-dependent properties of LA compounds with distinctive kinetic signatures such as lidocaine and bupivacaine.
, 百拇医药
Here we assayed the state-dependent accessibility of two cysteines engineered into the domain III, P-segment (μ1: F1236C, K1237C) to sulfhydryl modification with a methanethiosulphonate (MTS) reagent, MTS-ethylammonium (MTSEA). The rat skeletal muscle sodium channel (μ1) was used because this isoform has been well characterized especially with regard to slow inactivation and local anaesthetic block. Furthermore, previous studies have shown that these channels respond linearly to MTSEA reagents (Perez-Garcia et al. 1997). Using lidocaine and bupivacaine in a high-speed solution exchange system we are able to show, under identical pulse-train conditions, that sulfhydryl protection is conditionally dependent on the degree of use-dependent block. We propose that LA compounds may produce their kinetically distinct voltage-dependent behaviour by modulating slow inactivation gating to varying degrees.
, http://www.100md.com
Methods
Mutagenesis and heterologous expression
The rat skeletal muscle Na+ channel subunit (μ1) was subcloned into the HindIII–XbaI site of the vector green fluorescent protein with an internal ribosomal entry site (pGFPires) as previously described (Johns et al. 1997). Mutagenesis, F1236C or K1237C, was performed on the rat Na+ channel (Yamagishi et al. 1997) using a PCR-based method (QuickChange site-directed mutagenesis kit; Stratagene, La Jolla, CA, USA). All mutations were confirmed by dideoxynucleotide sequencing. Wild-type and mutated channels were expressed in HEK 293 cells using lipofectamine (Invitrogen Co., Carlsbad, CA, USA) along with Na+ channel 1 subunit (provided by Dr Alfred George, Vanderbilt University). For experiments with F1304Q (provided by Dr Gordon Tomaselli, Johns Hopkins University), cells were cotransfected with rat Na+ channel 1 subunit, and GFP. Transfected cells were incubated at 37°C in 95% O2–5% CO2 for 24–72 h before electrical recordings. Cells exhibiting green fluorescence were chosen for electrophysiological analysis.
, 百拇医药
Electrophysiology
All recordings were performed at room temperature (20–22°C). Whole-cell Na+ currents (INa) were obtained using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA) and were acquired using pCLAMP 8 (Axon Instruments). Patch pipettes were pulled from borosilicate glass (Warner Instrument Inc., Hamden, CT, USA) with a Model P-97 Flaming-Brown micropipette puller (Sutter Instruments, San Rafael, CA, USA). Pipette resistance was 1–3 M when filled with a pipette solution containing (mM): 140 NaF, 10 NaCl, 5 EGTA, 10 Hepes, pH 7.40. Replacing the intracellular K+ with Na+ eliminated the time-dependent K+ currents in our HEK cell recordings. For experiments involving F1304Q channels, pipette solution contained (mM): 10 NaF, 110 CsF, 20 CsCl, 10 EGTA, 10 Hepes, pH 7.35 with CsOH. The bath solution in both cases contained (mM): 150 NaCl, 4.5 KCl, 1.5 CaCl2, 1 MgCl2, 10 Hepes (pH 7.4 with NaOH). In all recordings, 80% of the series resistance was compensated, yielding a maximum voltage error of 1 mV. MTS reagents: MTSEA, MTS-ethylsulphonate (MTSES), and MTS-ethyltrimethylammonium (MTSET) (Toronto Research Chemicals, Toronto, Ontario, Canada) were kept on ice as high concentration stock solutions and were diluted to 200 μM, 300 μM, or 5 mM in 5 ml bath solution immediately before use. Lidocaine HCl (Sigma Chemical Co., St Louis, MO, USA) or bupivacaine HCl (Sigma Chemical Co.) were diluted from stock solutions to the bath concentrations indicated in the text. Cells were clamped in whole-cell mode for at least 10 min before recording data. Whole-cell currents were sampled at 20 kHz (DigiData 1200 A/D converter; Axon instruments) and low pass-filtered at 5 kHz.
, 百拇医药
Rapid solution exchange
Rapid solution exchange was achieved using a computer-controlled High Speed Solution Exchange system (HSSE) (ALA Scientific Instruments, Westbury, NY, USA). HSSE has two orthogonal microbore Teflon output tubes (300 μm inner diameter) which face each other at 90 deg in the same plane (as depicted in Fig. 1A). The pipette (along with the cell attached) was positioned close to the tips of the tubes. The solution flow through the two tubes was controlled using pCLAMP 8.0 software (Axon instruments). The time resolution of HSSE in our set-up was determined by exposing a high Na+ bath solution (150 mM NaCl) and a low Na+ bath solution (10 mM NaCl and 135 mM N-methyl-D-glucamine+) alternatively to wild-type Na+ channels using the protocol shown in Fig. 1B (inset). Briefly, a test pulse to –20 mV was applied in the presence of high Na+. After 50 ms at –120 mV the cell was exposed to the low Na+ solution for varying durations (P1). As seen in Fig. 1B, the direction of the Na+ current fully reversed by the time the cell was exposed to low Na+ for 30 ms, suggesting that solution exchange was completed within this time frame. The HSSE system was used only in experiments shown in Figs 4 and 5.
, 百拇医药
A, two orthogonal microbore tubes were positioned at right angles and in close proximity to the recording pipette and cell. Solution flow was switched between the tubes using signals from pCLAMP software. B, the minimum solution exchange time was determined using the protocol shown in the inset. Wild-type Na+ currents were measured using a double depolarization pulse protocol. The first pulse was applied in high Na+ solution, while the second pulse was applied in low Na+ solution after a variable P1 interval. Peak INa decreases and reverses direction within 30 ms after switching to low Na+ solution. C, voltage dependence of activation and inactivation of K1237C channels obtained using the protocols shown in the inset and fitted to a Boltzmann function as described in the text.
, http://www.100md.com
A, protocol used to examine the effects of use-dependent block on the MTSEA modification of K1237C. Peak INa was recorded during 100 repetitive pulses to –20 mV in the presence of lidocaine or bupivacaine and in the presence or absence of MTSEA. Repetitive pulses to –20 mV for 1 s were applied with a 3 s interpulse repolarization period (at –120 mV) to selectively allow recovery from lidocaine, but not bupivacaine block (see rectangle in Fig. 3A). B, in the absence of MTSEA, the data confirm nearly complete recovery from lidocaine block using a 3 s repolarization period (peak INa reduced only 10% even after 100 pulses), in contrast to bupivacaine (INa reduced 50% after 100 pulses). C, peak INa recorded in the presence of MTSEA using the protocol shown in panel A. Plotted are currents recorded before exposure to MTSEA (Pre) and 60 s after the pulse train (Post), overlaid for comparison. MTSEA was applied from 100 ms before the depolarization to 400 ms into the depolarization (a total of 500 ms) using the HSSE system, as indicated in the protocol shown in panel A. D, fractional modification by MTSEA is summarized in the bar graph as the percentage reduction of peak INa (1 – Post/Pre). The number of cells recorded in each condition is shown in parentheses. Enhanced slow inactivation in the presence of bupivacaine reduced modification of K1237C by MTSEA compared to lidocaine or control (P < 0.05).
, http://www.100md.com
A, development of LA block was evaluated using the protocol shown in the inset. Both 300 μM lidocaine () and 30 μM bupivacaine () produced similar levels of block of INa (shaded region) after prolonged depolarizations (500–1000 ms). B, peak INa recorded using the protocol shown in the panel. In both LAs 50 repetitive pulses were applied to –20 mV with a 3 s interpulse recovery at –120 mV. Here, MTSEA (5 mM) was applied for the latter 500 ms of each 1 s depolarization, using the HSSE system, to expose cells to MTSEA during similar LA block. Plotted are currents recorded before (Pre) and 60 s after pulse train (Post), overlaid for comparison. C, fractional modification by MTSEA is summarized in the bar graph as the percentage reduction of peak INa (1 – Post/Pre). The number of cells recorded in each condition is shown in parentheses. For similar levels of slow inactivation, bupivacaine and lidocaine protected K1237C residue from MTS modification to a similar extent (NS with each other but P < 0.05 with control).
, 百拇医药
Data analysis
The data were acquired and analysed using pCLAMP 8.0 software (Axon instruments). All results are expressed as means ± S.E.M. and statistical comparisons were made using one-way ANOVA (Origin, OriginLab Corp., Northampton, MA, USA) with P < 0.05 indicating significance. Multi-exponential functions were fitted to the data using nonlinear least-squares methods (Origin).
Results
We expressed rat skeletal muscle Na+ channel (μ1) in HEK-293 cells to allow whole-cell voltage-clamp measurements. We measured peak INa at varying activating voltages and after conditioning steps to a range of inactivating voltages (protocol inset, Fig. 1C). The voltage dependence of activation (open squares) and inactivation (filled squares) were plotted, and voltage-dependent parameters were derived by fitting a Boltzmann function:
, 百拇医药
where V1/1 is the half-maximal voltage and k is the slope factor. The V1/1 of activation was –31.6 ± 1.3 (n = 12) while the V1/1 of inactivation was –74 ± 0.7 (n = 10), consistent with previously published values (Chahine et al. 1996). Cummins et al. (1999) reported that HEK-293 cells have endogenous Na+ current with V1/1 of activation of –42.4 ± 3.2 mV and V1/1 of inactivation of –88.4 ± 1.9 mV. However, the values obtained from our experiments (see above) suggest that the current was predominantly due to the expressed rat skeletal muscle Na+ channels and not the endogenous channels.
, 百拇医药
Depolarization-dependent accessibility of pore cysteines to MTS reagents
To assess conformational changes in the pore, we first examined depolarization-dependent accessibility of pore cysteine mutations to sulfhydryl reagents. For comparison to our prior studies (Ong et al. 2000), we retested the accessibility of a cysteine engineered at the 1236 locus (F1236C), to sulfhydryl reagents using the same protocols from Ong et al. (Fig. 2A). Since prior studies (Perez-Garcia et al. 1997) showed that MTSEA modification reduced peak INa in a voltage-independent manner, all of our experiments were conducted with depolarizations to –20 mV, a potential at which peak inward current was maximum (data not shown). A 3 min exposure to 200 μM MTSEA during prolonged depolarization (protocol II) reduced F1236C peak sodium current (31 ± 3%) to a greater extent than hyperpolarization (protocol I) (20 ± 1%, P < 0.05). While these results reconfirm that depolarization increases the accessibility of the cysteine side chain (see Fig. 2 of Ong et al. 2000), they do not distinguish between changes in MTS reactivity due to fast or slow inactivation. The higher concentration of MTSEA required for depolarization-dependent modification of F1236C in this study was probably due to variation in the potency of the compound.
, 百拇医药
A, peak INa was recorded from HEK-293 cells transfected with F1236C or K1237C mutant channels during a control period, or in lidocaine (100 μM) or bupivacaine (10 μM). State-dependent modification of these cysteines by MTSEA was assessed with the two protocols (I and II) shown. After an initial pulse (P1) to –20 mV, MTSEA was washed into the bath. After a 1 min equilibration period, the membrane was hyperpolarized to –120 mV (protocol I) or depolarized to –20 mV (protocol II) for 3 min. After MTSEA was washed out (1 min), INa was reassessed using a brief pulse to –20 mV (P2). B, representative currents through K1237C channels elicited by P1 and P2 pulses are superimposed for comparison. The left and middle traces were obtained using protocol I and II, respectively. The trace on the right was obtained using protocol II in the presence of lidocaine. C, summary data showing the fractional reduction of peak INa due to sulfhydryl modification (1 – P2/P1). MTSEA was applied to the F1236C (200 μM) or K1237C (300 μM) mutants, respectively. Depolarization facilitated MTSEA modification of K1237C (P < 0.0001) and F1236C (P < 0.05), while MTSET or MTSES modification of K1237C was minimal with depolarization. Lidocaine and bupivacaine reduced the MTSEA modification of both F1236C and K1237C during depolarization (P < 0.05).
, http://www.100md.com
We next examined the accessibility of a cysteine at the adjacent 1237 position. Since K1237 is located in the deepest region of Na+ channel domain III P-loop, and forms part of the ion selectivity filter (DEKA) (Heinemann et al. 1992; Tsushima et al. 1997), we hypothesized that a cysteine in this position might be less accessible in the baseline (hyperpolarized) state, while maintaining a similar degree of accessibility as F1236C in the depolarized state. As seen in Fig. 2B, and summarized in the bar graph in Fig. 2C, exposure of MTSEA during depolarization reduced K1237C peak INa more than during hyperpolarization (31 ± 2% versus 15 ± 2%, P < 0.0001), and the difference was slightly greater than at the 1236 position (Fig. 2C). While the extent of MTSEA modification is consistent with our earlier studies (Ong et al. 2000), the gating-dependent differences in accessibility are consistently modest. This suggests that slow inactivation is associated with subtle, yet distinguishable, conformational changes in the pore. Identical experiments using the larger, positively charged reagent MTSET and the negatively charged analogue MTSES were performed, but neither reagent produced depolarization-dependent modification of INa (Fig. 2C). The insensitivity of K1237C to these MTS reagents (MTSET and MTSES) is consistent with a previous study (Chiamvimonvat et al. 1996).
, http://www.100md.com
As shown previously for the 1236 locus (Ong et al. 2000), lidocaine exposure attenuated the modification of K1237C by MTSEA during depolarization (Fig. 2B and C, 20 ± 3%, versus control depolarization, P < 0.05). For consistency with prior work, the effect of lidocaine to attenuate MTSEA modification of the 1236 locus was also reconfirmed (Fig. 2C). In addition, bupivacaine, a LA with far slower use-dependent recovery kinetics, also attenuated MTSEA modification of K1237C (Fig. 2C, 19 ± 2%, versus control depolarization, P < 0.05). These results indicate that inhibition of the depolarization-dependent MTSEA modification is not specific to lidocaine, but also holds true for other LA compounds.
, 百拇医药
Kinetic differences between local anaesthestics
To explore whether kinetic differences between LA compounds in use-dependent block correlate with cysteine side chain accessibility in the P-loop during gating, we first evaluated the rate at which K1237C channels recovered from block by either lidocaine or bupivacaine (Fig. 3A). The recovery kinetics were fitted to a biexponential function (Table 1), and the drug concentrations were selected to produce slow recovery components of similar magnitude (A2). Lidocaine (100 μM) and bupivacaine (30 μM) increased the amplitude of the slow recovery component to a similar degree (A2: 0.78 ± 0.01 for lidocaine, 0.79 ± 0.04 for bupivacaine). However, the time constant of recovery of the slow component (slow) in bupivacaine was much slower than in lidocaine (Fig. 3A: slow; 8744 ± 444 ms versus 314 ± 14 ms, P < 0.0001). The slower recovery from bupivacaine block is in agreement with previous findings (Clarkson & Hondeghem, 1985; Berman & Lipka, 1994).
, 百拇医药
A, recovery from inactivation and drug block in K1237C channels evaluated using the protocol shown in the inset of panel B. Continuous lines are two exponential fits to the mean data: y = y0 + A1 (1 – e–t/fast) + A2 (1 – e–t/slow). Recovery from inactivation was significantly delayed in 30 μM bupivacaine () compared to control () or 100 μM lidocaine (). Within 3 s, Na+ channels completely recovered from lidocaine block, while only 50% of the channels recovered from bupivacaine block. B, recovery from inactivation and drug block of fast inactivation deficient mutant channels, F1304Q. Inset shows INa recorded from F1304Q and K1237C channels. Note the removal of fast inactivation in F1304Q channels. As in K1237C, recovery from inactivation in F1304Q channels was significantly delayed in 30 μM bupivacaine () compared to control () or 100 μM lidocaine (). All parameters are defined and summarized in Table 1.
, http://www.100md.com
To determine if the distinctive slow recovery components induced by lidocaine and bupivacaine require intact fast inactivation gating structures, we utilized a previously described mutant Na+ channel lacking fast inactivation, F1304Q (Balser et al. 1996b). Figure 3B inset shows normalized INa recorded from K1237C and F1304Q channels during a step depolarization to –20 mV from a holding potential of –120 mV. While K1237C channels completely inactivate within a few milliseconds, F1304Q channels do not inactivate even after 10 ms, demonstrating that F1304Q channels are fast inactivation deficient. We examined the recovery from lidocaine and bupivacaine block of F1304Q (Fig. 3B). As expected, the magnitude of the fast recovery component (A1) is markedly reduced in drug-free conditions (Table 1). Nonetheless, in bupivacaine, the time constant of recovery of the slow component (slow) remains long, and far slower than in lidocaine (Fig. 3B: slow; 5088.5 ± 442.5 ms versus 167 ± 30.3 ms, P < 0.01). Moreover, recovery of the F1304Q slow component in lidocaine was still delayed compared to drug-free conditions (see Table 1). These results show that distinctive recovery components induced by these LA agents do not require intact fast inactivation, and corroborate the findings of Vedantham & Cannon (1999).
, 百拇医药
Slow inactivation protects MTS modification
Figure 3A also shows that 3 s after the end of the 1 s conditioning pulse, almost all K1237C channels had fully recovered (–120 mV) in the presence of lidocaine, while only 40–50% of channels recovered in the presence of bupivacaine (vertical bar, Fig. 3A). We made use of this difference to identify pulse train conditions that render cumulative block in bupivacaine, but not lidocaine. The protocol (Fig. 4A) applied repetitive 1 s depolarizing pulses to –20 mV with an interpulse repolarization to –120 mV for 3 s, allowing nearly complete recovery between pulses in lidocaine, but only partial recovery in bupivacaine. Figure 4B shows the representative current (every 10th pulse) in the presence of lidocaine or bupivacaine, in the absence of MTSEA. Peak INa is reduced by 50% at steady state in the presence of bupivacaine (right), but only by 10% in the presence of lidocaine (left), consistent with the distinctive use-dependent characteristics of the two agents, as also reflected in Fig. 3A.
, http://www.100md.com
We then examined whether marked differences in cumulative block by bupivacaine and lidocaine predictably track inactivation-dependent sulfhydryl modification of the 1237 cysteinyl. To selectively examine accessibility of the 1237 cysteinyl during inactivation, a rapid solution exchange system was utilized (Fig. 1). MTSEA was applied from 100 ms before the depolarization to 400 ms after depolarization (a total of 500 ms), as indicated in the protocol shown in Fig. 4A. While these exposure periods include both fast and slow inactivation gating components, our results in F1304Q (Fig. 3B) effectively exclude fast inactivation as the gating component primarily responsible for lidocaine–bupivacaine kinetic differences. In these experiments, the concentration of MTSEA was increased to 5 mM to allow substantial modification during these brief exposure periods. Since MTSEA was applied only during the period shown using the HSSE system, non-specific effects were minimized. Although depolarization in control, lidocaine, or bupivacine would lead to slow inactivation of Na+ channels during the initial 400 ms of each depolarization, we hypothesized that cumulative use-dependent bupivacaine block (under the conditions of the protocol in Fig. 4A) would ‘protect’ the K1237C residue from modification. In contrast, the complete recovery from inactivation in drug-free conditions or in lidocaine would allow MTS modification of unblocked channels at the onset of each depolarization. The measured currents, recorded before (Pre) and 60 s after the 100 depolarizing pulses (Post) allow for complete recovery from use-dependent drug block, so that any current reduction reflects MTS modification (superimposed in Fig. 4C). The data from all experiments are summarized in the bar graph (Fig. 4D), which shows the percentage of channels that have been modified by MTSEA. We measured less modification of K1237C by MTSEA in bupivacaine (39 ± 4%) than in lidocaine or control (52 ± 3% and 57 ± 3%, respectively, both P < 0.05 versus bupivacaine). This result suggests that accessibility of the 1237 cysteine to MTSEA is more restricted under kinetic conditions where use-dependent bupivacaine block is maintained, but use-dependent lidocaine block is not.
, 百拇医药
A gating-independent explanation for this result is that bupivacaine somehow provides better protection of the cysteinyl side chain, independent of its effect on inactivation gating. In this case, we would anticipate greater protection from MTSEA modification by bupivacaine under conditions where use-dependent INa reduction is similar to that evoked by lidocaine. Figure 5A shows the development of use-dependent block, in the absence or presence of lidocaine or bupivacaine, evaluated using the protocol shown in the inset. The concentrations of lidocaine (300 μM) and bupivacaine (30 μM) were chosen so that most of the channels (> 85%) were blocked by 500 ms or longer depolarizations to –20 mV (boxed region in Fig. 5A). To evaluate cysteine modification, a repetitive 1 s depolarizing pulse was applied, and MTSEA was selectively exposed for 500 ms in the latter portion of the 1 s depolarization pulse (Fig. 5B, upper panel), a period when most of the Na+ channels were blocked by lidocaine or bupivacaine (boxed region, Fig. 5A). As in Fig. 4, a 3 s interpulse repolarization to –120 mV was applied between each depolarization. The optimal approach would allow 1 min or more for full recovery of all channels from bupivacaine block (Fig. 3A); however, given the lengthy duration of the experiment, this was technically not feasible. While a 3 s interpulse period allowed only 40–50% of channels to recover from bupivacaine block (versus 100% in lidocaine), during all subsequent depolarizations, the channels are nonetheless fully blocked by either LA during the critical period (the latter 500 ms of Pn) when MTSEA is repeatedly exposed. Hence, the protocol (Fig. 5B) does provide conditions where MTSEA is exposed to channels when the extent of slow inactivation is saturated for lidocaine and bupivacaine, and thus similar, particularly in contrast to the conditions of Fig. 4.
, 百拇医药
The modification by MTSEA (Fig. 5B, bottom panel) was assessed before (Pre) and 60 s after the pulse train (Post). As before, this allowed for complete recovery from use-dependent lidocaine and bupivacaine block, so that any current reduction reflects MTS modification alone. Lidocaine significantly inhibited the modification of MTSEA compared to control (Fig. 5C: 17 ± 3% versus 27 ± 3%, P < 0.05), and the reduction of MTSEA modification by lidocaine was comparable to that of bupivacaine (14 ± 3%, P < 0.05 with control, NS versus lidocaine). Hence, similar degrees of use-dependent block have comparable effects on cysteinyl modification. The findings suggest that the differences found in Fig. 4D relate to drug-induced differences in gating (differential recovery from slow inactivation), as opposed to direct LA-specific interference of cysteine modification.
, http://www.100md.com
Discussion
Pore rearrangement during slow inactivation
Voltage-gated Na+ channels are dynamic molecules that modify their conformational state in response to changes in membrane potential. Depolarization leads to channel inactivation within a few milliseconds (fast inactivation), a process involving residues situated near the cytoplasmic face of the channel (West et al. 1992). Prolonged depolarization induces channels to progressively occupy more stable slow-inactivated states. Numerous studies have linked conformational changes in the pore-lining segments of Na+ channels to slow inactivation (Balser et al. 1996a; Kambouris et al. 1998; Mitrovic et al. 2000; Xiong et al. 2003). The same residues, particularly K1237C, play a critical role in ionic selectivity of the pore for Na+ over Ca2+ (Favre et al. 1996; Tsushima et al. 1997).
, 百拇医药
Here we show that slow inactivation alters the accessibility of a cysteine engineered at the 1237 locus to sulfhydryl modification (Fig. 2), supporting prior studies indicating the deepest region of Na+ channel pore has restrictive access and undergoes conformational rearrangement during slow inactivation (Benitah et al. 1999; Ong et al. 2000). Unlike the K1237E or K1237S (Todt et al. 1999), K1237C in the present experiment did not produce ‘ultra slow’ inactivation ( on the order of 100 s), but rather, exhibited normal recovery kinetics in the absence of MTSEA (Fig. 3A), suggesting cysteine substitution alone did not significantly alter the channel gating. A recent study (Struyk & Cannon, 2002) showed that MTSET accessibility to pore cysteine mutations C-terminal to the selectivity filter did not change during slow inactivation. In our studies, MTSET (or MTSES) accessibility to K1237C was also not affected during depolarization (Fig. 2C) consistent with a previous study (Chiamvimonvat et al. 1996). Hence, pore rearrangements during slow gating appear to be focused on the narrow selectivity filter locus (K1237C) and residues immediately N-terminal (F1236C) (Ong et al. 2000) that is not accessible to the larger MTSET or negatively charged MTSES.
, http://www.100md.com
Local anaesthetics and slow inactivation
LAs exert their therapeutic effects by suppressing the ionic currents through Na+ channels. While the efficacy of LA action is enhanced by membrane depolarization (use dependence), the mechanistic basis for compound-specific differences in use-dependent drug block remains debated. Enzymes and mutations that remove fast inactivation attenuate use-dependent suppression of sodium channels by lidocaine and other Na+ channel blockers (Cahalan, 1978; Yeh & Tanguy, 1985; Bennett et al. 1995), suggesting an interaction between LAs and the fast-inactivated state. However, recent studies have questioned the postulated linkage between fast inactivation and use-dependent block by demonstrating that structural components of the Na+ channel involved in fast inactivation gating are not trapped in the fast-inactivated state by lidocaine (Vedantham & Cannon, 1999). In the present work, removal of fast inactivation (using a fast inactivation deficient mutant) did not eliminate the slow component of recovery seen in lidocaine and bupivacaine. Moreover, lidocaine did not inhibit the covalent modification of 1237C when MTSEA was exposed during the initial 400 ms of depolarization, a period that includes fast inactivation (Fig. 4), further supporting the findings of Vedantham & Cannon (1999). At the same time, bupivacaine elicits a slowly recovering block component within this time frame (Fig. 3A), and protected the 1237 cysteine from MTSEA modification (Fig. 4C and D).
, http://www.100md.com
Using MTSEA modification of the adjacent cysteine (F1236C), we recently (Ong et al. 2000) showed that lidocaine attenuates MTSEA modification during sustained depolarizations, but not brief, 5 ms depolarizations, also suggesting that pore rearrangements linked to slow inactivation and use-dependent lidocaine action are mechanistically linked (Ong et al. 2000). In this study, we provide further insight into the relationship between the pore rearrangement and use-dependent LA action by leveraging the kinetic differences between lidocaine and bupivacaine. Specifically, these data suggest that pore conformational changes that alter MTSEA accessibility track the differences in rate of recovery from use-dependent block for the two LAs and implicate slow inactivation in the compound-specific differences in use-dependent block.
, http://www.100md.com
We postulate the conceptual model shown in Fig. 6. While K1237C is relatively inaccessible during hyperpolarization (Fig. 6, left), depolarization leads to conformational changes in the pore-lining segments that increase MTSEA accessibility (Fig. 2). LAs do not inhibit accessibility of MTSEA during opening or fast inactivation, presumably because LAs are not trapped in the pore (Fig. 6, middle) (Vedantham & Cannon, 1999). However, with the accumulation of use-dependent drug block, the pore rearranges such that LA binding is stabilized and MTSEA accessibility of K1237C is reduced. Recently Bai et al. (2003) reported that mutations in the S6-domain LA binding sites affect slow inactivation, strengthening the notion that the binding of LAs may stabilize a structural rearrangement related to slow inactivation. Moreover, our findings showing the recovery kinetics of bupivacaine and lidocaine track the MTSEA accessibility of the 1237 cysteine side chain (Fig. 4) suggest that the various LAs may stabilize the slow-inactivated conformational state to differing degrees. As such, measured differences in rate of recovery from use-dependent block may reflect differences in LA affinity for the rearranged pore (Fig. 6, right).
, 百拇医药
The 1237C side chain is relatively inaccessible in hyperpolarized channels (left). Depolarization leads to conformational changes that increase accessibility of the cysteine residue (K1237C) to MTSEA modification, during channel opening and fast inactivation (middle). Prolonged depolarization leads to slow inactivation, which is accompanied by pore rearrangement. LAs stabilize pore rearrangement during slow inactivation (right), resulting in the reduction in the rate of MTSEA modification (ka > kb). We postulate that the extent of stabilization is dependent on the distinctive binding kinetics of the two LAs, as the dissociation rate of bupivacaine from its binding site is likely to be slower than that of lidocaine (i.e. Kd,bupi < Kd,lid).
, 百拇医药
Sunami et al. (1997) have shown electrostatic interactions between lidocaine binding and K1237, suggesting that the native lysine lies in close proximity to the LA-binding site in domain IV, segment 6 (Ragsdale et al. 1994). At the same time, the 1236 and 1237 cysteine side chains are accessible to only extracellular application of MTSEA (Yamagishi et al. 1997), while the LAs block the skeletal muscle isoform (used here) only from inside the cell (Sunami et al. 2000), indicating that a physical interaction whereby the LAs directly inhibit MTSEA activity across the selectivity filter is unlikely. Studies have shown that diverse drugs, including LAs, bind to the same residues in the transmembrane IV S6 segment of voltage-gated Na+ channels (Ragsdale et al. 1996). Mutation of these binding residues have distinct effects on different drugs, suggesting that the compounds may interact with the S6 domain in an overlapping but non-identical manner, which could depend on their chemical structure. It has also been postulated that LAs interact with hydrophobic aromatic residues (phenylalanine or tyrosine) through hydrophobic or electron interactions (Ragsdale et al. 1994). In this regard, bupivacaine has two aromatic rings while lidocaine has only one. Moreover, three hydrophobic aromatic residues face the pore lumen in the putative S6 LA binding site (Ragsdale et al. 1994). Since bupivacaine has two aromatic rings, it is possible that electron interactions are enhanced, and stabilize slow inactivation, thus delaying recovery from drug block. We postulate that LAs stabilize structural rearrangements coupled to slow inactivation by interaction with pore-lining side chains in or near the selectivity filter, and that use-dependent kinetic differences between LAs may partly result from differences in the stability of this drug-bound conformational state.
, http://www.100md.com
References
Adelman WJ Jr & Palti Y (1969). The effects of external potassium and long duration voltage conditioning on the amplitude of sodium currents in the giant axon of the squid, Loligo pealei. J General Physiol 54, 589–606.
Bai CX, Glaaser IW, Sawanobori T & Sunami A (2003). Involvement of local anesthetic binding sites on IVS6 of sodium channels in fast and slow inactivation. Neurosci Lett 337, 41–45.
, 百拇医药
Balser JR, Nuss HB, Chiamvimonvat N, Perez-Garcia MT, Marban E & Tomaselli GF (1996a). External pore residue mediates slow inactivation in mu1 rat skeletal muscle sodium channels. J Physiol 494, 431–442.
Balser JR, Nuss HB, Orias DW, Johns DC, Marban E, Tomaselli GF & Lawrence JH (1996b). Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels. J Clin Invest 98, 2874–2886.
, 百拇医药
Bean BP, Cohen CJ & Tsien RW (1983). Lidocaine block of cardiac sodium channels. J General Physiol 81, 613–642.
Benitah JP, Chen Z, Balser JR, Tomaselli GF & Marban E (1999). Molecular dynamics of the sodium channel pore vary with gating: interactions between P-segment motions and inactivation. J Neurosci 19, 1577–1585.
Bennett PB, Valenzuela C, Chen LQ & Kallen RG (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. Circ Res 77, 584–592.
, 百拇医药
Berman MF & Lipka LJ (1994). Relative sodium current block by bupivacaine and lidocaine in neonatal rat myocytes. Anesth Analg 79, 350–356.
Cahalan MD (1978). Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons. Biophys J 23, 285–311.
Chahine M, Deschene I, Chen LQ & Kallen RG (1996). Electrophysiological characteristics of cloned skeletal and cardiac muscle sodium channels. Am J Physiol 271, H498–H506.
, 百拇医药
Chen Z, Ong BH, Kambouris NG, Marban E, Tomaselli GF & Balser JR (2000). Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels. J Physiol 524, 37–49.
Chiamvimonvat N, Perez-Garcia MT, Ranjan R, Marban E & Tomaselli GF (1996). Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis. Neuron 16, 1037–1047.
Choi KL, Aldrich RW & Yellen G (1991). Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc Natl Acad Sci U S A 88, 5092–5095.
, http://www.100md.com
Clarkson CW & Hondeghem LM (1985). Mechanism for bupivacaine depression of cardiac conduction: fast block of sodium channels during the action potential with slow recovery from block during diastole. Anesthesiology 62, 396–405.
Courtney KR (1975). Mechanism of frequency-dependent inhibition of sodium currents in frog myelinated nerve by the lidocaine derivative GEA. J Pharmacol Exp Ther 195, 225–236.
Cummins TR, Dib-Hajj SD, Black JA, Akopian AN, Wood JN & Waxman SG (1999). A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons. J Neurosci 19, RC43.
, 百拇医药
Favre I, Moczydlowski E & Schild L (1996). On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel. Biophys J 71, 3110–3125.
Heinemann SH, Terlau H, Stuhmer W, Imoto K & Numa S (1992). Calcium channel characteristics conferred on the sodium channel by single mutations. Nature 356, 441–443.
Hilber K, Sandtner W, Kudlacek O, Glaaser IW, Weisz E, Kyle JW, French RJ, Fozzard HA, Dudley SC & Todt H (2001). The selectivity filter of the voltage-gated sodium channel is involved in channel activation. J Biol Chem 276, 27831–27839.
, 百拇医药
Hille B (1977). Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J General Physiol 69, 497–515.
Hodgkin AL & Huxley AF (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117, 500–544.
Hoshi T, Zagotta WN & Aldrich RW (1991). Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. Neuron 7, 547–556.
, 百拇医药
Johns DC, Nuss HB & Marban E (1997). Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs. J Biol Chem 272, 31598–31603.
Kambouris NG, Hastings LA, Stepanovic S, Marban E, Tomaselli GF & Balser JR (1998). Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat micro1 skeletal muscle sodium channel. J Physiol 512, 693–705.
, 百拇医药
Lopez-Barneo J, Hoshi T, Heinemann SH & Aldrich RW (1993). Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1, 61–71.
Mitrovic N, George AL Jr & Horn R (2000). Role of domain 4 in sodium channel slow inactivation. J General Physiol 115, 707–718.
Ong BH, Tomaselli GF & Balser JR (2000). A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence. J General Physiol 116, 653–662.
, http://www.100md.com
Perez-Garcia MT, Chiamvimonvat N, Ranjan R, Balser JR, Tomaselli GF & Marban E (1997). Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification. Biophys J 72, 989–996.
Ragsdale DS, McPhee JC, Scheuer T & Catterall WA (1994). Molecular determinants of state-dependent block of Na+ channels by local anesthetics. Science 265, 1724–1728.
Ragsdale DS, McPhee JC, Scheuer T & Catterall WA (1996). Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels. Proc Natl Acad Sci U S A 93, 9270–9275.
, http://www.100md.com
Struyk AF & Cannon SC (2002). Slow inactivation does not block the aqueous accessibility to the outer pore of voltagegated Na channels. J General Physiol 120, 509–516.
Sunami A, Dudley SC Jr & Fozzard HA (1997). Sodium channel selectivity filter regulates antiarrhythmic drug binding. Proc Natl Acad Sci U S A 94, 14126–14131.
Sunami A, Glaaser IW & Fozzard HA (2000). A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel. Proc Natl Acad Sci U S A 97, 2326–2331.
, 百拇医药
Todt H, Dudley SC Jr, Kyle JW, French RJ & Fozzard HA (1999). Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule. Biophys J 76, 1335–1345.
Tsushima RG, Li RA & Backx PH (1997). Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore. J General Physiol 109, 463–475.
Vedantham V & Cannon SC (1999). The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels. J General Physiol 113, 7–16.
, http://www.100md.com
Wang GK, Brodwick MS, Eaton DC & Strichartz GR (1987). Inhibition of sodium currents by local anesthetics in chloramine-T-treated squid axons. The role of channel activation. J General Physiol 89, 645–667.
West JW, Patton DE, Scheuer T, Wang Y, Goldin AL & Catterall WA (1992). A cluster of hydrophobic amino acid residues required for fast Na+-channel inactivation. Proc Natl Acad Sci U S A 89, 10910–10914.
Xiong W, Li RA, Tian Y & Tomaselli GF (2003). Molecular motions of the outer ring of charge of the sodium channel: do they couple to slow inactivation J General Physiol 122, 323–332.
, 百拇医药
Yamagishi T, Janecki M, Marban E & Tomaselli GF (1997). Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis. Biophys J 73, 195–204.
Yeh JZ & Tanguy J (1985). Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons. Biophys J 47, 685–694.
Yellen G, Sodickson D, Chen TY & Jurman ME (1994). An engineered cysteine in the external mouth of a K+ channel allows inactivation to be modulated by metal binding. Biophys J 66, 1068–1075., http://www.100md.com(Koji Fukuda, Tadashi Naka)