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1Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana; 2Department of Anesthesiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; and 3Department of Biology, State University of New York, Albany, New York
Submitted 5 April 2006; accepted in final form 26 June 2006
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ABSTRACT |
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INTRODUCTION |
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). However, it is not clear how TFP is able to induce analgesia. TFP is one of the most potent calmodulin (CaM) inhibitors among the phenothiazine neuroleptics (Brostrom and Wolff 1981; Levin and Weiss 1979). CaM, a 16.7-kDa protein that is expressed in virtually all eukaryotic cells, can bind calcium and induce changes in target proteins. Several studies indicate that CaM can bind to and alter the activity of voltage-gated sodium channels (Deschenes et al. 2002; Tan et al. 2002), and we previously showed that CaM can modulate Nav1.4 (skeletal muscle) and Nav1.6 (neuronal) current amplitudes and properties (Herzog et al. 2003). TFP is also able to reduce peak sodium current in a concentration-dependent manner in giant squid axons (Ichikawa et al. 1991). These studies raise the possibility that TFP could exert its analgesic effects by modulating sodium currents in mammalian neurons and that this might involve disruption of the CaM-channel interaction.
Alterations in sodium currents within the peripheral nervous system can contribute to changes in cellular excitability and are likely to play important roles in nociception. The predominant sodium channel isoforms in nociceptive neurons are Nav1.7, Nav1.8, and Nav1.9. Although the C-terminal region of sodium channels that contains the CaM binding motif is well conserved, our previous results indicated that the C termini of Nav1.8 and Nav1.9 do not appreciably bind CaM (Herzog et al. 2003). The C terminus of Nav1.7 does bind CaM, although it has a lower apparent affinity than the C termini of other TTX-sensitive isoforms. Nav1.7 (also referred to as PN1) is expressed at high levels in the peripheral nervous system (Sangameswaran et al. 1997; Toledo-Aral et al. 1997), and deletion of the mouse Nav1.7 gene in a subset of sensory neurons that are predominantly nociceptive decreases responses to noxious mechanostimulation and induced inflammatory pain (Nassar et al. 2004). Furthermore, it has been recently reported that patients with a painful inherited neuropathy, primary erythermalgia, have mutations in their Nav1.7 channels that cause significant hyperpolarizing shifts in the channel V1/2 of activation as well as larger ramp currents (Cummins et al. 2004; Yang et al. 2004). Because of its important role in nociception, modulation of Nav1.7 currents could be a factor in the analgesic effects of TFP.
Although sodium channels are subject to extensive modulation, little is known about modulation of Nav1.7. It has been recently reported that Nav1.7 expressed in Xenopus oocytes can be modulated by protein kinase A and protein kinase C (Vijayaragavan et al. 2004). However, it is not known if CaM, or CaM antagonists, can modulate Nav1.7 currents. The goal of the present study was to investigate if TFP can modulate Nav1.7 current and if this modulation was due to inhibition of the channel interaction with CaM. As our previous results indicated that Nav1.7 had a lower affinity for CaM than Nav1.4 channels (Herzog et al. 2003), we predicted that if TFP could modulate Nav1.7 current by altering the interaction with CaM, then TFP would have dissimilar effects on Nav1.4 and Nav1.7. Understanding the effects of TFP on Nav1.7 could lead to a better understanding the drug's analgesic properties and possible use in regional anesthesia and thus pain management.
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METHODS |
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HEK293 cells were grown under standard tissue culture conditions (5% CO2; 37°C) in DMEM supplemented with 10% fetal bovine serum. Transfections of Nav1.4 and Nav1.7 were performed using the calcium phosphate precipitation technique. No 
subunits were transfected with the channels as previous studies have shown that subunits do not alter the effect of CaM on sodium currents in HEK293 cells (Deschênes et al. 2002; Young and Caldwell 2005). The calcium phosphate-DNA mixture was added to the cell culture medium and left for 15–20 h, after which time the cells were washed with fresh medium. After 48 h, antibiotic (G418, Geneticin; Cellgro, Herndon, VA) was added to select for neomycin-resistant cells. After 2–3 wk in G418, colonies were picked, split, and subsequently tested for channel expression using whole cell patch-clamp recording techniques.
Chemicals and solutions
For application during voltage-clamp experiments, TFP (Sigma Aldrich, St. Louis, MO) was dissolved in sterile water to give stock solutions of 10 mM and 300 µM, respectively. Calmodulin inhibitory peptide (EMD Biosciences, San Diego, CA) was dissolved in sterile water to give a stock solution of 240 µM. Lidocaine hydrochloride was purchased from Sigma Aldrich. For the sciatic nerve blockade, TFP at 10, 20, 40, and 80 mM and lidocaine at 40 mM were dissolved in 0.9% sodium chloride shortly before the experiments. On local injection, the relatively low pH of these solutions (pH range, 3.9–6.0) is likely to be buffered quickly by the tissue fluid, which has a pH of 7.4.
Whole cell patch-clamp recordings
Whole cell patch-clamp recordings were conducted at room temperature (
21°C) using a HEKA EPC-10 double amplifier. Data were acquired on a Windows-based Pentium IV computer using the Pulse program (v 8.65, HEKA Electronic). Fire-polished electrodes (0.9–1.3 M
) were fabricated from 1.7-mm capillary glass using a Sutter P-97 puller (Novato, CA). The standard pipette solution contained (in mM) 140 CsF, 10 NaCl, 1.1 EGTA, and 10 HEPES, pH 7.3. The pipette solution used for the calmodulin inhibitory peptide experiments contained (in mM) 90 CsF, 10 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), 10 NaCl, 10 HEPES, pH 7.3. CsOH was used to adjust pH for the pipette solutions. The standard bathing solution contained (in mM) 140 NaCl, 1 MgCl2, 3 KCl, 1 CaCl2, and 10 HEPES, pH 7.3 (adjusted with NaOH). Series resistance errors were compensated to be <3 mV.
Subfascial sciatic nerve injections
The protocol for animal experimentation was reviewed and approved by the Harvard Medical Area Standing Committee on Animals (Boston, MA). Female Sprague-Dawley rats were purchased from Charles River Laboratory (Wilmington, MA) and kept in the animal housing facilities at Brigham and Women's Hospital with controlled relative humidity (20% - 30%), at room temperature (24°C), and in a 12-h (6:00 AM to 6:00 PM) light-dark cycle. Rats were handled before the procedure to familiarize them with the experimental environment and to minimize stress-induced analgesia. At the time of injections, animals weighed 200–250 g and showed no signs of neurobehavioral impairment. The experimenter was blinded to the drug and concentration used. After rats were anesthetized by inhalation of 1–2% isoflurane, the sciatic nerves were exposed by lateral incision of the thighs and division of the superficial fascia and muscle. The test dose (0.2 ml) was injected directly beneath the clear fascia surrounding the nerve but outside the perineurium, proximal to the sciatic bifurcation, with a 30-gauge needle attached to a tuberculin syringe. The test doses comprised of TFP at concentrations from 10 to 80 mM, lidocaine at 40 mM as active control, and normal saline (vehicle only), n = 8 per group. The superficial muscle layer was sutured with 4–0 silk, and the wound was closed as described (Kalichman et al. 1989).
Neurobehavioral examination
The neurobehavioral examination, modified from earlier reports (Thalhammer et al. 1995), focused on motor and sensory function. Initially, rats were examined at 30 min and 1 h after drug administration, then at 1-h intervals until 3 h and at 6, 12, 24, and 48 h. Briefly, we evaluated motor function by measuring the extensor postural thrust of the hind limbs by holding the rat upright with the hind limb extended so that the distal metatarsus and toes support the animal's weight, thereby measuring the extensor thrust as the gram force applied to a digital platform balance (Ohaus Lopro, Fisher Scientific, Florham Park, NJ). The reduction in this force, representing reduced extensor muscle contraction due to motor blockade, was calculated as a percentage of the control force (preinjection control value range was 125–155 g). The percentage value was assigned a score: 0 = no block or baseline; 1 = minimal block, force between 100 and 50% of preinjection control value; 2 = moderate block; force between 50% of the preinjection control value and 20 g (20 g is the weight of the flaccid limb); 3 = complete block, force equal to or <20 g.
Sensory function (pain withdrawal) was evaluated by the withdrawal reflex or vocalization to pinch of a skin fold over the lateral metatarsus (cutaneous pain) and of the distal phalanx of the fifth toe (deep pain). This nocifensive reaction was graded in the following manner on a scale of 0–3 and based on withdrawal reflex, escape behavior, and vocalization: 0 (baseline or normal, brisk withdrawal reflex, normal escape behavior and strong vocalization), 1 (mildly impaired), 2 (moderately impaired), and 3 (totally impaired nocifensive reaction). We repeated the examination three times; the average was used.
Data analysis
Voltage-clamp experimental data were analyzed using the Pulsefit (v 8.65, HEKA Electronic) and Origin (OriginLab, Northhampton, MA) software programs. Slope factors of steady-state inactivation curves were calculated using the general Boltzmann function: I(V) = offset + {amplitude/1 + exp[–(V – Vhalf)/slope]}. Two-way ANOVA was applied to test for differences in motor sciatic nerve block duration and sensory block duration among four different doses of TFP and 40 mM lidocaine. A significant F test was followed by multiple pairwise group comparisons using the post hoc Tukey procedure. To protect against type I errors (false positives), a power analysis indicated that the sample size of eight animals per group provided 80% statistical power ( = 0.2, 2-tailed 
= 0.05) to detect significant differences in block duration using ANOVA among the four doses of TFP and in the TFP versus lidocaine comparison (version 6.0, nQuery Advisor, Statistical Solutions, Boston, MA). We presented the data in all figures in terms of the mean and SE because we tested the data at each dose within the drug groups for normality using the Kolmogorov-Smirnov goodness-of-fit statistic and found no significant departures from a normal distribution (P > 0.10 in each case). Because this check on normality was verified, we then chose to report means ± SEs and analyze the data parametrically using ANOVA. Error bars in the figures represent the SE. Statistical analysis was performed using the SPSS software package (version 14.0, SPSS, Chicago, IL). All reported P values are two-tailed.
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RESULTS |
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To determine if TFP could affect mammalian voltage-gated sodium currents, TFP was applied externally to HEK293 cells stably transfected with human Nav1.7 or human Nav1.4, and sodium currents were monitored using whole cell voltage-clamp techniques. Cells were held at –100 mV and pulsed to 0 mV every 5 s to elicit channel current. In the presence of 500 nM or 2 µM TFP, sodium currents rapidly declined and stabilized within 15 and 25 s for Nav1.7 and Nav1.4, respectively (Fig. 1, A and C). Although a small hyperpolarizing shift in the current-voltage relationship for Nav1.7 and Nav1.4 was observed after application of 2 µM TFP (Fig. 1, B and D), these shifts were not significantly different from the time-dependent shifts observed in control cells. Nav1.7 showed decreases in peak sodium currents of 19.4 ± 6.0% (n = 6) and 76.7 ± 4.6% (n = 6) after 2.5 min during application of 500 nM and 2 µM TFP, respectively. Under the same conditions, the same two concentrations of TFP produced decreases in peak Nav1.4 currents of 14.7 ± 7.3% (n = 6) and 59.4 ± 7.8% (n = 5), respectively.
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To understand how TFP reduces sodium current amplitude, we investigated the effect of TFP on steady-state inactivation. We observed that application of 500 nM and 2 µM TFP to cells containing Nav1.7 caused hyperpolarizing shifts of –5.0 ± 0.87 mV (n = 6) and –18.4 ± 2.3 mV (n = 6) in the V1/2 of steady-state inactivation, respectively (Fig. 2, A and B), where V1/2 was determined by fitting the data with the standard Boltzmann function as shown in METHODS. The slope factor of the steady-state inactivation curve for Nav1.7 in the absence of TFP was 6.6 ± 0.1. After application of 500 nM and 2 µM TFP, the slope factors were 7.0 ± 0.3 and 10.2 ± 0.5, respectively. Application of the same two concentrations of TFP to cells containing Nav1.4 caused hyperpolarizing shifts in the V1/2 of steady-state inactivation of –5.0 ± 0.87 mV (n = 5) and –11.4 ± 0.80 mV (n = 6), respectively (Fig. 2, D and E). The slope factor of the steady-state inactivation curve for Nav1.4 in the absence of TFP was 6.0 ± 0.1. After application of 500 nM and 2 µM TFP, the slopes were 5.9 ± 0.1 and 6.6 ± 0.3, respectively. In control experiments (without TFP application), time-dependent shifts were observed in the V1/2 of steady-state inactivation taken 7 min apart for Nav1.7 channels (–2.6 ± 0.43 mV; n = 6) and Nav1.4 channels (–4.7 ± 1.1 mV; n = 5). Thus application of 500 nM TFP did not cause a significant hyperpolarizing shift in the V1/2 of steady-state inactivation of Nav1.7 or Nav1.4 compared with the hyperpolarizing shift that was observed in these channels over the same period of time in control experiments. However, 2 µM TFP did cause a significant hyperpolarizing shift in the V1/2 of steady-state inactivation of Nav1.7 channels and Nav1.4 channels compared with time-dependent controls (Fig. 2, C and F).
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The hyperpolarizing shifts of steady-state inactivation of both Nav1.7 and Nav1.4 during TFP application indicates that TFP enhances sodium channel inactivation and led us to propose that TFP might exhibit use-dependent inhibition of sodium channels. We investigated this by pulsing the transfected cells to –10 mV at a frequency of 5 Hz in the absence and presence of drug. Figure 3 (A and B) displays examples of results under control conditions (left) and in the presence of 2 µM TFP (right) for both Nav1.7 and Nav1.4. In the presence of 500 nM TFP, there were 67.6 ± 3.3% (n = 6) and 54.1 ± 6.7% (n = 5) decreases from the current amplitudes evoked by the first pulse to the current amplitudes evoked by the last pulse in the protocol for Nav1.7 and Nav1.4, respectively. In the presence of 2 µM TFP, decreases of 73.8 ± 5.6% (n = 6) and 80.4 ± 3.4% (n = 6) were observed for Nav1.7 and Nav1.4, respectively. All of these decreases in peak current amplitudes were significant compared with time-dependent decreases in peak current amplitudes in the absence of drug (Fig. 3, C and D).
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To determine if the mechanism by which TFP altered Nav1.7 and Nav1.4 involved inhibition of the CaM-sodium channel interaction, currents were observed in the presence of a more specific calmodulin inhibitor. For these experiments, we used calmodulin inhibitory peptide, a 17-residue peptide based on the calmodulin-binding domain of myosin light chain kinase that binds to calmodulin with a Kd of 6 pM (Torok et al. 1998). This inhibitor is not cell permeable and had to be added directly to the pipette solution. Recordings were taken 12 min after obtaining whole cell configuration to allow for adequate diffusion of the peptide into the cell.
Changes in steady-state inactivation of Nav1.7 were observed in the presence of 1 µM calmodulin inhibitory peptide (Fig. 4A). The mean V1/2 of steady-state inactivation of Nav1.7 in the presence of 1 µM calmodulin inhibitory peptide was significantly hyperpolarized compared with without the peptide (Fig. 4C). In contrast, steady-state inactivation of Nav1.4 was unaffected by the presence of 1 µM calmodulin inhibitory peptide (Fig. 4B). Although there is a small difference, the mean V1/2 of inactivation of Nav1.4 in the presence and absence of the peptide were not statistically different (Fig. 4C). These results revealed that specific inhibition of CaM had significant effects on Nav1.7 steady-state inactivation while having no effect on Nav1.4 steady-state inactivation.
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We also examined whether calmodulin inhibitory peptide induced use-dependent inhibition of voltage-gated sodium currents. Figure 5, A and B, displays examples of the protocol results in the presence of 1 µM calmodulin inhibitory peptide for Nav1.7 and Nav1.4, respectively. In cells recorded with 1 µM calmodulin inhibitory peptide added to the pipette solution the use-dependent decrease of Nav1.4 current amplitudes was small (12.43 ± 1.69%; n = 5) but significantly larger compared with the use-dependent decrease in cells recorded in pipette solution without the peptide (4.19 ± 0.19%; n = 5, Fig. 5C). Calmodulin inhibitory peptide (1 µM) in the pipette solution induced a small but significant use-dependent effect on currents produced by Nav1.7 stimulated at high-frequency (Fig. 5C). Nav1.7 stimulated at high frequency with the pipette solution alone showed a 16.6 ± 5.1% (n = 5) decrease in current. This decrease was significantly smaller than the 32.4 ± 4.3% (n = 5) decrease in current seen with 1 µM calmodulin inhibitory peptide in the pipette solution (Fig. 5C). The use-dependent effects of the CaM inhibitory peptide on Nav1.7 and Nav 1.4, although significant, were not as large as the effects seen with TFP. This result, in conjunction with our observation that the calmodulin inhibitory peptide had no effect on Nav1.4 steady-state inactivation, led us to hypothesize that the effects of TFP seen on these channels could be the result of something other than blocking CaM-channel interactions.
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As local anesthetics (LA) are known to exhibit use-dependent inhibition of sodium currents and induce a negative shift in the voltage dependence of sodium channel steady-state inactivation, we hypothesized that TFP might be acting as a LA. According to the modulated receptor hypothesis of LA inhibition of sodium channels, the affinity of the LAs for sodium channels depends on the state of the channel with inactivated channels exhibiting a much higher affinities than resting channels. We therefore tested the ability of 2 µM TFP to block Nav1.7 and Nav1.4 channels in the resting state by using a holding potential of –140 mV. Current was elicited with a pulse to 0 mV every 5 s, and TFP caused a 8.3 ± 0.5% (n = 10) and 8.4 ± 1.1% (n = 10) decrease in Nav1.7 and Nav1.4 current, respectively. This indicates that block of resting channels by TFP is similar for Nav1.7 and Nav1.4, with an estimated IC50 of 22 µM. To examine TFP binding to inactivated channels, cells were held at –120 mV and prepulsed to –60 mV for 10 s, pulsed to –120 mV for 50 ms to allow for recovery from fast inactivation, and then pulsed to 0 mV to elicit current. With this protocol, TFP caused a 85.6 ± 2.1% (n = 5) and 87.4 ± 8.2% (n = 5) decrease in Nav1.7 and Nav1.4 current, respectively. This indicates that block of inactivated channels by TFP is also similar for Nav1.7 and Nav1.4, with an estimated IC50 of 300 nM. These data suggest that, like LA inhibition, TFP inhibition is state dependent. This also indicates that the disparity in block of Nav1.7 and Nav1.4 currents by 2 µM TFP observed in Fig. 1 can be explained by differences in steady-state availability rather than differences in channel affinities for TFP.
TFP increases the onset of inhibition and slows recovery from inhibition for Nav1.7 and Nav1.4
We further investigated the effect of TFP by examining the time course for onset of inhibition and recovery from inhibition for Nav1.7 and Nav1.4 currents. Figure 6A (top) displays the protocol used to examine the onset of TFP inhibition for Nav1.7 and Nav1.4 currents. The onset of inhibition for Nav1.7 and Nav1.4 currents by 2 µM TFP was rapid compared with the development of slow inactivation observed under control conditions (Fig. 6A, bottom). The time constants for the inhibition of Nav1.7 and Nav1.4 currents by 2 µM TFP were 49.1 ± 3.9 ms (n = 13) and 62.6 ± 6.0 ms (n = 14), respectively, and were not significantly different. The onset of Nav1.7 and Nav1.4 current inhibition by 500 nM TFP was significantly slower giving time constants of 148 ± 24 ms (n = 5) and 139 ± 26 ms (n = 5), respectively. Figure 6B displays the protocol used to examine the recovery from TFP inhibition for Nav1.7 and Nav1.4 currents. The recovery of Nav1.7 currents from inhibition by 500 nM and 2 µM TFP (Fig. 6C, top) had time constants of 572 ± 36 µs (n = 6) and 686 ± 43 µs (n = 9), respectively. The recovery of Nav1.4 currents from inhibition by 500 nM and 2 µM TFP (Fig. 6C, bottom) was slower with time constants of 1.0 ± 0.1 s (n = 5) and 1.2 ± 0.06 s (n = 10), respectively. TFP concentration did not have a significant effect on the time constants of recovery for either Nav1.7 or Nav1.4. This is consistent with the interaction seen between LAs and sodium channels in which drug unbinding rate was unrelated to drug concentration, indicating a simple bimolecular reaction of drug and channel (Bean et al. 1983). To confirm that TFP was acting as a LA on Nav1.7 or Nav1.4, we tested the effects of TFP on Nav1.4 channels that have mutations in their LA binding site.
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LAs can directly bind to voltage-gated sodium channels and block the propagation of action potentials. Previous studies have shown that point mutations of residues located in the S6 transmembrane segments of domain 1 (DI), domain 3 (DIII), and domain 4 (DIV) of voltage-gated sodium channels can reduce high-affinity binding of LAs to the inactivated state of the channel (Li et al. 1999; Nau et al. 1999, 2003; Ragsdale et al. 1994; Wright et al. 1998). Furthermore, it has been shown that the tricyclic antidepressant amitriptyline, which blocks voltage-gated sodium channels with a high degree of additional use-dependent block under repetitive pulses, also interacts with the LA binding site on sodium channels (Barber et al. 1991; Nau et al. 2000). Three specific S6 residues located at DI, DIII, and DIV have been characterized as crucial for both LAs and amitriptyline binding in rat skeletal muscle (Nav1.4) sodium channels (Wang et al. 2004). Lysine and arginine substitutions at residues N434 (DI–S6), L1280 (DIII–S6), and F1579 (DIV-S6) have been shown to reduce the affinity for both amitriptyline and LA binding to the inactivated state of the channel (Nau and Wang 2004; Wang et al. 2004). To determine if TFP was acting through a possible LA mechanism, we applied TFP to rat Nav1.4 channels having one of three amino acid substitutions (N434K, L1280K, F1579K) and looked for changes in effectiveness of TFP inhibition.
Because these LA binding-site mutations were performed with rat Nav1.4 and the data shown in Figs. 1–3 used human Nav1.4, the control conditions involved application of TFP to wild-type rat Nav1.4. Rat and human Nav1.4 channels are highly conserved, and completely conserved in the CaM and LA binding site regions. Figure 7A displays the effects of application of 2 µM TFP to the external bath solution on sodium currents from the wild-type rat Nav1.4 and the LA binding site mutant channels. The addition of 2 µM TFP to wild-type rat Nav1.4 caused a decrease in peak sodium current of 83.7 ± 2.6% (n = 6; Fig. 7B). Two of the LA binding site mutant channels, N434K and F1579K, showed a significant attenuation in the decrease of peak sodium current caused by 2 µM TFP to 12.5 ± 1.8% (n = 5) and 7.01 ± 3.2% (n = 5), respectively. In contrast, the L1280K mutant channel showed a decrease of 80.3 ± 3.1% (n = 5) in peak sodium current which was not significantly different from the decrease observed with the wild-type channel (Fig. 7B).
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Use-dependent effects of TFP were also studied with the rat Nav1.4 as well as with the LA binding-site mutant channels by using the same 5-Hz pulse protocol that was used with the human Nav1.7 and Nav1.4. Decreases in current evoked by the first pulse to current evoked by the last pulse of 84.6 ± 1.3% (n = 11) for the wild-type rat Nav1.4 was observed in the presence of 2 µM TFP (Fig. 7D). All three of the LA binding-site mutant channels showed significantly attenuated use-dependent decreases in current produced by high-frequency stimulation in the presence of 2 µM TFP compared with the wild-type rat Nav1.4 channel (Fig. 7D). The decreases observed in the presence of 2 µM TFP were 44.1 ± 1.7% (n = 5), 23.3 ± 5.4% (n = 5), and 35.2 ± 5.0% (n = 6) for LA binding-site mutant channels N434K, L1280K, and F1579K, respectively (Fig. 7D). However, the attenuated use-dependent decrease observed for the L1280K channel might not accurately reflect a decreased sensitivity of the channel to use-dependent inhibition brought on by TFP as 2 µM TFP decreased the steady-state L1280K current to the point at which additional use-dependent inhibition was difficult to determine. Therefore we also examined current produced from the LA binding-site mutant channels and the wild-type channel after high-frequency stimulation in the presence of 500 nM TFP in an attempt to limit the initial effects on the L1280K channel. Our results show that 500 nM TFP produced a use-dependent decrease in current evoked by the first pulse to current evoked by the last pulse of 72.1 ± 2.7% (n = 8) for the wild-type rat Nav1.4 channel. The decreases observed for LA binding-site mutant channels N434K, L1280K, and F1579K were 14.7 ± 2.2% (n = 6), 33.2 ± 3.8% (n = 6), and 15.1 ± 1.9% (n = 8), respectively. All of these observed use-dependent decreases were significantly attenuated compared with the use-dependent decreases seen with the wild-type rat Nav1.4 channel.
Interestingly, the steady-state inactivation curves for each of the local anesthetic binding site mutant channels, in the absence of TFP, show that the N434K and F1579K mutations shifted steady-state inactivation of the channel in the depolarizing direction while the L1280K mutation shifted it in the hyperpolarizing direction compared with the wild-type channel (Fig. 8). Therefore with a holding potential of –100 mV, more of the L1280K channels are in the inactivated state than the N434K or F1579K channels, and this raises the possibility that the differential effect of TFP on the mutant channels could be influenced by the differential shifts in steady-state inactivation and may not reflect differences in state-dependent binding. To address this, we used protocols specifically designed to examine TFP binding to either the inactivated or closed state of the channels.
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140 nM. The estimated IC50s for TFP on the inactivated state of the N434K, L1280K, and F1579K channels are 1.6 µM, 250 nM, and 1.2 µM, respectively. Although all mutant channels had more peak current remaining compared with the peak current remaining in the wild-type rat Nav1.4, only the LA binding-site mutant channels N434K and F1579K showed a significance difference from the wild-type channel.
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27 µM. LA mutant channels N434K and F1579K after 2 µM TFP application had 103.9 ± 2.5% (n = 6) and 97.4 ± 3.8% (n = 8) of their current remaining, respectively, and these effects were not significantly different from those seen with the wild-type rat Nav1.4 (Fig. 9B). The L1280K mutant channel, however, showed a significant decrease in percent of remaining current in the presence of 2 µM TFP with only 41.6 ± 3.3% (n = 5) remaining (Fig. 9B). The estimated IC50s for TFP on the closed state of the L1280K and F1579K channels are 1.0 and 75 µM, respectively. The IC50 for TFP on the closed state of the N434K mutant channel could not be estimated due to the amount of current remaining after TFP treatment being >100%. However, it is probable that the IC50 value is >75 µM. Thus although the N434K and F1579K mutations reduced the apparent affinity of inactivated Nav1.4 for TFP, the L1280K mutation increased the apparent affinity of Nav1.4 in the closed state for TFP.
To further investigate the possibility that these lysine substitutions directly affect TFP binding rather than indirectly attenuating TFP inhibition by altering the inactivation of the untreated channel, we examined the effects of TFP on the development of channel inhibition as well as on recovery from inhibition. Surprisingly, the N434K channels showed an enhanced (2.5 times) development of inhibition by 2 µM TFP compared with WT channels while the F1579K channels showed a slower (12 times) development of inhibition by 2 µM TFP (Table 1). Based on the rates of channel inhibition by TFP observed for WT and N434K channels, inactivating prepulses used for examining recovery from channel inhibition by TFP were set at 200 ms. However, based on the rate of channel inhibition, a 200-ms prepulse would not be long enough to allow for adequate drug-binding to F1579K channels, and therefore a 500-ms inactivating prepulse was used for examining recovery of F1579K from inhibition by TFP. Table 1 shows that the N434K and F1579K channels recover from 2 µM TFP inhibition 4.5 and 3 times faster than do WT channels, respectively. These results indicate that while the N434K enhances the onset of TFP binding to inactivated Nav1.4, the F1579K mutation attenuates the onset of TFP binding to the channel, and both mutations accelerate TFP unbinding from the inactivated Nav1.4. These results strengthen the evidence that the differential effect of TFP on the mutant channels are due to differences in direct binding of the drug to the channel.
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Based on the in vitro effects of TFP on Nav1.7 and Nav1.4, we wanted to determine the extent of TFP's effects in vivo compared with the traditional local anesthetic lidocaine. We therefore performed subfascial injections of TFP and lidocaine to sciatic nerves in anesthetized rats and analyzed block of motor and sensory function after recovery. No block was seen in the normal saline (vehicle-only) group, all other rats developed a dose-dependent degree of sciatic nerve blockade after subfascial injections of TFP (n = 8 per group). All rats recovered completely with no clinically detectable neurologic deficits, except a slight motor deficit of rats in the 80 mM TFP group.
Two-way ANOVA indicated overall group differences in duration of motor sciatic nerve block [Fig. 10A, F(4,35) = 110.38, P < 0.0001]. Specific pairwise group comparisons with the post hoc Tukey procedure revealed that the sciatic nerve blockade was significantly different for 80 versus 40 mM TFP (P < 0.0001), 40 versus 20 mM TFP (P < 0.0001), and 20 versus 10 mM TFP (P = 0.024). In general, a highly significant dose-response relationship showed a less rapid return to baseline for the higher test doses. In addition, two-way ANOVA with repeated measures indicated that the blockade at test dose of 40 mM TFP was significantly different from using the same dose of lidocaine (P < 0.0001). Similarly, a two-way ANOVA indicated overall group differences in duration of sensory nerve block [Fig. 10B, F(4,35) = 49.51, P < 0.0001]. Multiple pairwise group comparisons with the Tukey procedure revealed that the sensory blockade was significantly different for 80 versus 40 mM TFP (P < 0.0001) and between 40 versus 20 mM TFP (P = 0.001). No significant difference in sensory blockade duration was observed between 20 versus 10 mM TFP (P = 0.57). ANOVA indicated that the sensory blockade at test dose of 40 mM TFP was also significantly different from using the same dose of lidocaine (P < 0.001).
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DISCUSSION |
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), we show that TFP decreases current amplitude for both Nav1.7 and Nav1.4 and causes hyperpolarizing shifts in Nav1.7 and Nav1.4 steady-state inactivation while having no effect on the voltage dependence of activation. We demonstrate that TFP has use-dependent inhibition effects on these channels and provide evidence that the predominant inhibitory effect of TFP is through binding to the LA binding site of these channels. Our data indicate that only a minor effect of TFP is likely due to preventing CaM interaction with the channels. We further reveal the mechanism of TFP inhibition by identifying specific amino acid residues that are involved in the binding of TFP to closed and inactivated Nav1.4 channels. Finally, we showed that TFP can cause sensory and motor blockade in vivo after subfascial injection to rat sciatic nerve. Our initial hypothesis for this work was that blocking CaM interactions with the IQ motif in the C terminus of these channels by using TFP would influence the gating of the channels. However, the Nav1.4 currents, which were dramatically inhibited by TFP, were largely unaffected by the specific CaM inhibitor, calmodulin inhibitory peptide, at high concentrations (1 µM; KD = 6 pM). The calmodulin inhibitory peptide did show significant effects on Nav1.7 steady-state inactivation, but the use-dependent effects were much smaller than those induced by TFP. This indicated to us that, while CaM could play a role in regulating steady-state inactivation of Nav1.7, the more profound effect of TFP on Nav1.7 might be independent of CaM inhibition, suggesting that TFP altered sodium currents through a different mechanism. Therefore we investigated an alternative mechanism for the effects of TFP on Nav1.7 and Nav1.4.
Previous studies have reported that several phenothiazine neuroleptics that are structurally related to TFP can also block sodium currents in a use-dependent manner (Ahmmed et al. 2002; Bolotina et al. 1992; Liu et al. 1992; Ogata et al. 1990). However, the ability of TFP and these other neuroleptics to directly interact with sodium channels, and the molecular mechanism of this possible drug-channel interaction, has not previously been determined. LAs are known to produce hyperpolarizing shifts in steady-state inactivation and use-dependent inhibition of sodium currents by binding to a receptor site on the ion channel (Bean et al. 1983; Hille 1977). These studies and our observations of TFP causing hyperpolarizing shifts in steady-state inactivation, exhibiting a higher apparent affinity for inactivated channels and exhibiting a concentration independent recovery from inhibition led us to hypothesize that TFP might interact with Nav1.7 and Nav1.4 through the LA receptor. The attenuation of the inhibitory effects of TFP on the N434K and F1579K mutant channel currents indicates that these residues are important for the interaction of TFP with sodium channels and that TFP acts directly on the channel. Surprisingly, the inhibitory effects of TFP on L1280K mutant channel current were greater than (Fig. 7A) or equal to (Fig. 7B) the effects on wild-type Nav1.4. Although this is different from the effect that the L1280K mutation has on LA and amitriptyline binding, it is consistent with the hypothesis that the TFP binding site and the LA binding site overlap.
Our results show that TFP preferentially binds to the inactivated state of voltage-gated sodium channels compared with the closed state. The N434K and F1579K mutant Nav1.4 channels showed a significantly decreased mean response to 500 nM TFP in their inactivated state, indicating that the binding of TFP to the channel is reliant on these two residues when the channel is inactivated. Interestingly, the L1280K channel showed no significant difference in the apparent affinity of the inactivated state. However, inhibition of the L1280K channel in the closed state was potentiated compared with that of the wild-type, N434K, and F1579K channels. This result indicates that the L1280K mutation enhances the binding of TFP to the closed-state of the channel. Overall, our data show that Nav1.4 in its inactivated state has a higher affinity for TFP, and the N434 and F1579 residues are important for efficient binding of TFP to the inactivated state of the sodium channel. Furthermore, these results show that the L1280 residue has less involvement in the binding of TFP to the inactivated state of the channel than residues N434 and F1579. However, these results also show that out of the N434, L1280, and F1579 residues, only the L1280 residue is important for TFP binding to the closed-state of voltage-gated sodium channels.
Due to conserved homology between the Nav1.4 and Nav1.7 LA binding site, we hypothesize that the inhibitory effects of TFP on human Nav1.7 are due to direct interaction with this site. Sciatic nerve block was chosen to examine in vivo effects as TFP given intrathecally cannot be extrapolated to provide analgesia (and anesthesia) in peripheral nerves due to peripheral nerves nerve fibers being myelinated and embedded in multiple layers of relatively thick nerve sheaths. Our data suggest that the direct interaction with neuronal sodium channels could help explain the finding that TFP injections in sciatic nerve produces motor and sensory blockade. The nerve block by TFP was found to be robust and more potent than lidocaine suggesting that TFP or TFP analogues might be useful for inducing prolonged nerve block.
Overall, our experiments show that the inhibition of sodium current caused by the CaM inhibitor TFP is largely independent of the inhibition of CaM interactions. The majority of the inhibition by TFP can be attributed to direct interaction of the drug with the sodium channels in a state-dependent manner. Interestingly, TFP's label as a potent CaM inhibitor has resulted in its effects being commonly related to inhibition of the calmodulin/CaMKII pathway. Studies using TFP to inhibit CaM and its effectors could be overlooking the direct action of TFP on sodium channels. This inhibitory action might lead to inaccurate conclusions regarding CaM's involvement in physiological processes. More specifically, studies correlating CaM inhibition with nerve block using TFP could be demonstrating a direct interaction of the drug with the voltage-gated sodium channels that are highly expressed in peripheral nerves. The ability of TFP to block Nav1.7 at low doses could make it a potential therapeutic regimen for regional anesthesia and pain management that would be more potent than traditional local anesthetics.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Cummins, Dept. of Pharmacology and Toxicology, Indiana University School of Medicine, 950 W. Walnut St., R2 468, Indianapolis, IN, 46202 (E-mail: trcummin{at}iupui.edu)
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