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CELLULAR AND MOLECULAR

Inhibition of the A-Type K⁺ Channels of Dorsal Root Ganglion Neurons by the Long-Duration Anesthetic Butamben

Departments of Pathology, Anatomy, and Cell Biology (D.L.B.W., M.E.O.) and Biochemistry and Molecular Pharmacology (C.L.B.), Jefferson Medical College, Philadelphia, PA; and Department of Physiology, Leiden University Medical Center, Leiden, The Netherlands (D.L.B.W., D.L.Y.)

Received April 11, 2005; accepted May 19, 2005.

	Abstract

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n-Butyl-p-aminobenzoate (BAB; butamben) is a long-duration anesthetic used for the treatment of chronic pain. Epidural administration of BAB is thought to reduce the electrical excitability of dorsal root nociceptor fibers by inhibiting voltage-gated ion channels. To further investigate this mechanism, we examined the effects of BAB on the potassium currents of acutely dissociated neurons from the rat dorsal root ganglion (DRG). These neurons express a rapidly inactivating A-type K⁺ current (I_A) that is resistant to tetraethylammonium (20 mM) but inhibited by 4-aminopyridine (5 mM). At low concentrations, BAB (1 µM) selectively inhibited the I_A component of DRG K⁺ current. The voltage dependence of activation and inactivation, kinetics of recovery from inactivation, and the pharmacology of the DRG I_A were similar to those of the Kv4 family of K⁺ channels. Reverse transcription-polymerase chain reaction was used to establish that the messages encoding for all three of the mammalian Kv4 channel subunits (Kv4.1–Kv4.3) were present in the rat DRG. BAB produced a high-affinity, partial inhibition of heterologously expressed Kv4.2 channels (K_D = 59 nM) but did not alter the kinetics or voltage sensitivity of gating. Substituting polar threonines for conserved hydrophobic residues of the S6 segment weakened BAB binding but did not alter the voltage-dependent gating of the Kv4.2 channel. At physiological pH, BAB is uncharged, suggesting that hydrophobic interactions may contribute to drug binding. The data support a mechanism in which BAB binds near the narrow cytoplasmic entrance of Kv4 channels and inhibits current by a pore blocking mechanism.

Although the long-duration anesthesia produced by epidural BAB is well documented, the underlying mechanism remains unclear. BAB is an inhibitor of the sodium, calcium, and delayed rectifier potassium currents of DRG neurons (Van den Berg et al., 1996; Beekwilder et al., 2003, 2005), suggesting that the reduced electrical excitability of dorsal root pain fibers is the most likely mechanism of BAB anesthesia (Van den Berg et al., 1995). Electrophysiological characterization of small DRG neurons has identified three broad classifications of voltage-gated K⁺ current expressed in these cells: a fast-inactivating ( 10-ms) 4-aminopyridine (4-AP)-sensitive A-type current (I_A), a slowly inactivating ( 100-ms) current, and a tetraethylammonium (TEA)-sensitive sustained current (Kostyuk et al., 1981; Christian et al., 1994; Gold et al., 1996; Safronov et al., 1996; Fedulova et al., 1998; Yoshimura and de Groat, 1999; Sculptoreanu et al., 2004). Studies of the voltage dependence, gating kinetics, and pharmacology have provided insight into the properties of the different components of DRG K⁺ current but have not yet identified the channels that underlie these currents. Immunochemical studies suggest that numerous voltage-gated K⁺ channels (Kv) are expressed in DRG neurons (Kv1 and Kv2) (Ishikawa et al., 1999). This is in agreement with the presence of RNA transcripts encoding for Kv1, Kv2, and Kv4 subunits in the DRG (Kim et al., 2002; Park et al., 2003; Yang et al., 2004). The data indicate that multiple Kv channels contribute to the K⁺ current important for setting the resting membrane potential and for repolarizing DRG neurons after an action potential.

In this study, we investigated the effects of BAB on the rapidly inactivating A-type K⁺ current (I_A) of small DRG neurons. The voltage-dependent gating and pharmacology of the endogenous K⁺ currents were similar to that of Kv4 channels, suggesting that these channels may contribute to the A-type current expressed in DRG neurons. BAB was found to be a potent inhibitor of both the DRG I_A and Kv4 currents. These findings indicate that the BAB inhibition of DRG Kv4 channels may contribute to the long-duration anesthesia produced by the epidural administration of this drug.

	Materials and Methods

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Isolation of DRG Neurons. Seven-day-old rats were sacrificed, and dorsal root ganglia from all accessible levels of the spinal cord were collected. The ganglia were mechanically dissociated in a coated (poly-L-lysine, mol. wt. 70,000–150,000; Sigma-Aldrich, St. Louis, MO) culture dish containing F-12 Ham's media (Kainghn's modification; Sigma-Aldrich) enriched with 1 mM CaCl₂, 2 mM glutamine, 30 mM NaHCO₃, 40 mM glucose, and 1% penicillin-streptomycin. Cells were allowed to attach to polylysine-coated culture dishes for 2.5 h in a humidified 5% CO₂ atmosphere at 37°C. Thereafter, the media were exchanged for similar culture media fortified with 10% horse serum. The whole-cell K⁺ currents of small DRG neurons (20 µm, 8–12 pf) were recorded within 8 h of isolation. Animals were euthanized in accordance with the Animal Welfare protocols of Thomas Jefferson University.

Expression of Kv4 Channels in tsA201 Cells. Details about the cDNA constructs of Kv4.2 and Kv4.3 have been published previously (Blair et al., 1991; Serodio et al., 1996). A standard calcium phosphate precipitation procedure was used to transfect tsA201 cells, a transformed human embryonic kidney 293 cell line (O'Leary and Horn, 1994). Cells were cotransfected with cDNA (5 µg) encoding for the Kv4 channel and CD8 cell surface marker (5 µg). The cDNA was added to 0.5 ml of 250 mM CaCl₂ and slowly mixed with 0.5 ml of 2x HeBS solution consisting of 275 mM NaCl, 40 mM HEPES, 12 mM dextrose, 10 mM KCl, and 1.4 mM Na₂HPO₄, pH 7. The mixture was incubated for 20 min at room temperature and then slowly added to a 100-mm culture dish of 50% confluent tsA201 cells in 10 ml of Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) enriched with 10% fetal bovine serum and 1% penicillin-streptomycin. After 3 h, the cells were washed and replated on 35-mm culture dishes. Transfected tsA201 cells expressing the CD8 marker display rapidly inactivating A-type K⁺ current (5–20 nA). This contrasts with nontransfected tsA201 cells, which have endogenous K⁺ currents of <100 pA.

Electrophysiology. Whole-cell patch recordings were made using Sylgard-coated (Dow Corning, Midland, MI) patch electrodes fashioned from Corning 8161 glass (Wilmad Glass Company, Buena, NJ). Series resistance was less than 1 M and was 80% compensated, resulting in voltage-clamp errors of <5 mV. Currents were filtered at 5 kHz and recorded using an Axopatch 200A amplifier and pClamp software (Axon Instruments Inc., Burlingame, CA). Internal solutions consisted of 140 mM KCl, 1 mM MgCl₂, 5 mM EGTA, and 10 mM HEPES titrated to pH 7.4 with NaOH for tsA201 cells or 140 mM KMeSO₄, 5 mM EGTA, and 10 mM HEPES titrated to pH 7.4 with NaOH for DRG neurons. External solutions consisted of 145 mM NaCl, 2 mM KCl, 1.5 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES titrated to pH 7.4 with NaOH for tsA201 cells or 140 mM choline, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES titrated to pH 7.4 with NaOH for DRG neurons. Control and drug solutions were continuously applied using a perfusion pipette positioned approximately 100 µm from the cell. BAB was dissolved in ethanol, resulting in bath concentrations of ethanol of 1%, which was included in drug-free control solutions. This concentration of ethanol had no discernible effect on Kv4 current. The bath temperature was maintained at room temperature (22°C) using a Medical Systems TC-202 temperature controller.

Site-Directed Mutagenesis. Site-directed mutations of the Kv4.2 channel (accession no. NM031730.1) were made using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) according to the supplier's instructions. Base substitutions were confirmed by automated DNA sequencing (Nucleic Acid Facility, Jefferson Medical College, Philadelphia, PA). Primers used to make these mutations were as follows: V397T, 5'-GAGCGGAGTCTTGACCATCGCGCTACCCGTGC and 5'-CGATCACAGGCACGGGTGTCGCGATGACCAAG; L400T, 5'-CTTGGTCATCGCGACACCCGTGCCTGTGATCG and 5'-CGATCACAGGCACGGGTGTCGCGATGACCAAG; and V404T, 5'-CGCGCTACCCGTGCCTACGATCGTGTCTAACTTC and 5'-GAAGTTAGACACGATCGTAGGCACGGGTAGCGCG.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The total RNA of the rat DRG was isolated using RNaqueous-4PCR (Ambion, Austin, TX) according the supplier's instructions. The extracted RNA (0.1 µg) was used as a template for RT-PCR reactions (SuperScript One-Step RT-PCR; Invitrogen) using gene-specific primers for Kv4 channels (Kv4.1, 5'-CGGACAAATGCTGTGCGTTAG-3', 5'-TAGGGGAGGAAGGTTGACTTTCAT-3'; Kv4.2, 5'-GACGTGAGGGGACAGAGAAC-3', 5'-CCCCACTTCTTCACCTCAGA-3'; and Kv4.3, 5'-TGACAACACTGGGGTATGGA-3', 5'-AACAGGGGATCATCCACAAG-3'). cDNA was synthesized by incubating the reaction mixture at 50°C for 30 min before polymerase chain reaction amplification. The cDNA was then denatured at 94°C for 2 min, followed by 35 cycles at 94°C for 15 s, 55°C for 30 s, and 72°C for 15 s. The amplified products were resolved on 2% agarose gels containing ethidium bromide (0.5 µg/ml). The specificity of the polymerase chain reaction reactions was evaluated by omitting the reverse transcriptase from the reaction mixture, which consistently eliminated cDNA amplification. The amplified cDNA bands were excised from the agarose gels (Qiaquick extraction kit; QIAGEN, Valencia, CA) and sequenced (Nucleic Acid Facility, Jefferson Medical College). The predicted size of the cDNA amplicons and the sequences were consistent with those previously published for Kv4 channels.

	Results

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Characterization of the DRG A-Type (I_A) K⁺ Current. Small DRG neurons (20 µm) typically display a variable combination of rapidly inactivating A-type (I_A), slowly inactivating, and sustained components of K⁺ current (Kostyuk et al., 1981). Figure 1A shows the whole-cell current of a DRG neuron expressing all three of these currents. External application of 10 mM TEA selectively reduced the current near the end of the depolarizing pulse consistent with the preferential inhibition of the delayed rectifier K⁺ current in these cells (Akins and McCleskey, 1993; Christian et al., 1994; Gold et al., 1996). Increasing the concentration to TEA from 10 to 20 mM resulted in a small additional reduction in the residual current and no change in the peak amplitude (data not shown). At +20 mV, the decay of the current measured in the presence of TEA was found to be biexponential with fast and slow time constants of 24.7 ± 2.2 and 128.9 ± 17.2 ms, respectively, with the rapid component accounting for 39.3 ± 0.03% of the current under these conditions (n = 18). By contrast, Fig. 1B shows the current of a DRG neuron predominately expressing the slowly inactivating and sustained components. In these cells, TEA produced a nearly equivalent reduction in the amplitude of the peak and sustained currents. A-type current was frequently detected in these cells but was difficult to study because of the small amplitude and functional overlap with TEA-resistant K⁺ currents expressed in these neurons. Figure 1C shows a DRG neuron expressing a large component of rapidly inactivating A-type current (I_A). The I_A component was relatively insensitive to TEA but was potently inhibited by 5 mM 4-AP, which is an effective inhibitor of A-type K⁺ current (Gustafsson et al., 1982; Thompson, 1982).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Whole-cell K+ currents measured from small (20 µm) acutely dissociated rat DRG neurons. Currents were elicited by a depolarizing test pulse (20 mV/400 ms) from a holding potential of –65 mV. A 2-s conditioning pulse to –80 mV was applied immediately before the test pulse to reduce steady-state inactivation. A, cell displaying a combination of inactivating A-type (IA) and sustained K+ current. B, neuron predominately expressing slowly inactivating and sustained K+ currents. C, neuron expressing a large component of rapidly inactivating A-type current. Currents are shown before (control) and after the bath application of 10 mM TEA or 5 mM 4-AP.

To further investigate the properties of the DRG I_A, we selected cells expressing a prominent component of A-type current similar to that shown in Fig. 2A. The voltage-dependent activation of the I_A component was determined by measuring the peak current amplitude elicited by depolarizing test pulses. The conductance at each voltage was calculated, normalized to the conductance determined at +40 mV, and plotted versus the test voltage (Fig. 2B). The DRG I_A began to activate at voltages more depolarized than –60 mV, suggesting that the channels underlying this current may be active near the resting membrane potential (–55 mV) of these neurons (Christian et al., 1994; Wang et al., 1997; Sculptoreanu et al., 2004). The steady-state availability was determined from conditioning pulses between –120 and 0 mV and plotted versus voltage (Fig. 2B). The smooth curves are fits to a Boltzmann function with a midpoint of –74 mV. The recovery from inactivation was determined by applying a depolarizing conditioning pulse to inactivate the channels before measuring the recovery time course at –90 mV (Fig. 2C). The smooth curve is a fit to an exponential function with a time constant of 107.6 ± 7.1 ms (n = 4), consistent with the rapid recovery of I_A at hyperpolarized voltages.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Functional characterization of the DRG A-type current. A, whole-cell DRG K+ current were elicited by depolarizing to voltages between –80 and +40 mV from a holding potential of –65 mV. A hyperpolarizing prepulse (–80 mV/2 s) was applied immediately prior to the test protocols to minimize steady-state inactivation. B, conductance (G) at each voltage was calculated G = Ip/(Vt – Vr), where Ip is peak the current amplitude, Vt the test pulse voltage, and Vr the reversal potential (–85 mV). G was normalized to that determined at +40 mV (Go) and plotted versus the test voltage. The smooth curves are fits to a fourth-order Boltzmann function (G/Go = 1/(1 + exp[–(Vr – Vm)/k 4])) with an apparent threshold (Vm) and slope factor (k) of –60.3 ± 10.1 and 29.6 ± 5.8 mV (n = 7). Also plotted is the steady-state inactivation determined by applying 900-ms prepulses to voltages between –120 and 0 mV. A standard test pulse (20 mV/400 ms) was used to assess availability. The test currents were normalized to that measured after prepulsing to –120 mV and plotted versus voltage. Noninactivating current was removed by subtracting the persistent current measured after prepulsing to 0 mV. The smooth curves are fits to a Boltzmann function [h = 1/(1 + exp(V – V0.5)/k)] with midpoint (V0.5) and slope factor (k) of –74.0 ± 1.1 and 14.0 ± 1.0 mV (n = 6). C, recovery from inactivation was determined by applying a depolarizing conditioning pulse (20 mV/400 ms) before returning the voltage to –90 mV for a variable duration (0–1000 ms). A standard test pulse (20 mV/400 ms) was used to monitor the recovery of the channels. The test currents were normalized to that measured after full recovery (–90 mV/1000 ms) and plotted versus the recovery interval. The smooth curve is a fit to an exponential function with time constant and residual noninactivating current of 107.6 ± 7.1 ms and 0.21 ± 0.03, respectively (n = 4).

Comparison of the DRG A-Type and Heterologously Expressed Kv4 Currents. The DRG I_A displays subthreshold activation, fast inactivation, and rapid recovery from inactivation at hyperpolarized voltages. Members of the Shaker (Kv1.4), Shaw (Kv3.3 and Kv3.4), and Shal (Kv4.1, Kv4.2, and Kv4.3) subfamilies of vertebrate K⁺ channels produce A-type current similar to the DRG I_A (Coetzee et al., 1999). Kv1.4 channels display rapid voltage-dependent activation and inactivation, are insensitive to TEA, and blocked by millimolar concentrations of 4-AP similar to what we observed for the DRG A-type current (Fig. 1). An important distinguishing feature is the time course of recovery from inactivation of Kv1.4, which is slow ( = 2–4 s) by comparison with what we observed for the DRG I_A ( = 107 ms). Kv3 channels are inhibited by submillimolar concentrations of externally applied TEA (Coetzee et al., 1999), which is inconsistent with our finding that the I_A component is not appreciably inhibited by comparatively high concentrations (10–20 mM) of this blocker (Fig. 1). By contrast, Kv4 channels are relatively insensitive to TEA, inhibited by 4-AP, display subthreshold activation, and rapidly recovery from inactivation at hyperpolarized voltages (Jerng et al., 2004), properties that are similar to what we observed for the DRG I_A (Fig. 1).

We directly compared the biophysical properties of the DRG I_A with those of heterologously expressed Kv4 channels. Figure 3A shows the whole-cell K⁺ current of a tsA201 cell expressing Kv4.2. The K⁺ conductance at each voltage was determined from the peak current amplitude, normalized to the conductance measured at +40 mV, and plotted versus the test potential (Fig. 3B). The threshold for Kv4.2 activation (V_m =–39.8 mV) was shifted toward depolarized voltages by comparison with the native A-type current of DRG neurons (V_m = –60.3 mV). The steady-state availability was determined for voltages between –95 and 0 mV and plotted versus the prepulse potential (Fig. 3B). The smooth curve is a fit of the data to a Boltzmann function with V_0.5 of –63.4 mV (Table 1). The recovery from inactivation was determined by applying depolarizing prepulses to inactivate the channels before measuring the recovery time course at –100 mV (Fig. 3C). The recovery from inactivation was well fitted with an exponential function with a time constant of 82.1 ± 4.2 ms (n = 4). At +20 mV, the decay of the Kv4.2 current was biexponential with fast and slow time constants of 13.4 ± 1.5 and 87.8 ± 4.6 ms, respectively (n = 5). With the exception of a depolarizing shift in the midpoint of inactivation, the steady-state properties of the heterologously expressed Kv4.3 channel were similar to those of Kv4.2 (Table 1). Despite some differences in the voltage dependence, the gating of the DRG I_A component resembles that of heterologously expressed Kv4 channels, suggesting that these channels may contribute to the rapidly inactivating K⁺ current in these neurons.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Functional properties of heterologously expressed Kv4.2 channels. A, current was activated by depolarizing to voltages between –60 and +40 mV from a holding potential of –100 mV. B, conductance at each voltage was determined as described previously (Fig. 2), normalized to the conductance at +40 mV, and plotted versus voltage. The smooth curve is a fit of the data to a fourth-order Boltzmann function with an apparent threshold (Vm) and slope factor (k) of –39.2 ± 2.3 and 20.7 ± 1.3 mV (n = 5). Also plotted is the steady-state inactivation determined by applying 900-ms prepulses to voltages between –95 and 0 mV before assaying availability using a standard test pulse (0 mV/200 ms). The test currents were normalized to those measured at –95 mV and plotted versus prepulse potential. The smooth curve is a fit of the data to a Boltzmann function with V0.5 and k of –63.4 ± 0.1 and 4.6 ± 0.1 mV (n = 4), respectively (Table 1). The residual noninactivating component of Kv4.2 current in these experiments was 2.6 ± 0.3%. C, recovery from inactivation of Kv4.2 was measured by applying a depolarizing prepulse (+10 mV/400 ms) to inactivate the channels before applying a variable duration (1–600 ms) hyperpolarization to –100 mV. The fractional recovery at various times was assessed using a standard test pulse (+10 mV/75 ms). The recovery time course was exponential with a time constant of 82.1 ± 4.2 ms (n = 4).

To further investigate the contribution of Kv4 channels to the DRG K⁺ current, we used RT-PCR to determine whether the RNA transcripts encoding for the Kv4 channels was present in the rat DRG. A single band corresponding to nonconserved regions of the Kv4.1, Kv4.2, and Kv4.3 subunits was amplified from DRG RNA (Fig. 4, RT+). No bands were detected when reverse transcriptase was omitted (RT–) from the reaction mixture confirming that DNA amplification is dependent on the transcription of RNA. The RT+ bands were sequenced and found to be identical to the published cDNA sequences of the respective Kv4 subunits. The presence of transcripts encoding for Kv4 channel subunits in the rat DRG further supports the conclusion that Kv4 channels contribute to the A-type current of these neurons.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Characterization of Kv4 channel expression in rat DRG by RT-PCR. Total RNA extracted from the rat DRG was used as the template for RT-PCR reactions. Gene-specific primers for each of the Kv4 subunits were used to amplify cDNA, and the products were resolved on agarose gels. The gel shows parallel reactions for each of the Kv4 channel primers in which the reverse transcriptase was omitted (RT–) or included (RT+) in the reaction mixture. The sizes of the cDNA amplicons are consistent with those predicted for Kv4.1 (467 base pairs), Kv4.2 (600 base pairs), and Kv4.3 (400 base pairs) channels. The amplified products in the RT+ lanes were extracted from the gel and further analyzed by automated DNA sequencing. The end lanes were a 100-base pair cDNA ladder.

View larger version (18K): [in this window] [in a new window]   Fig. 5. BAB inhibition of DRG IA and Kv4.2 channels. A, DRG IA current elicited by depolarizing test pulses (20 mV/400 ms) before and after application of 1 µM BAB. BAB significantly inhibited the IA component of DRG K+ current 12.6 ± 0.5% (n = 4) by comparison with drug-free controls (paired t test; p < 0.05). B, heterologously expressed Kv4.2 currents shown before (control) and after application of BAB (1 µM). BAB significantly reduced the peak current amplitudes by 27.2 ± 1.0% (paired t test; p < 0.005; n = 4). C, peak Kv4.2 currents measured after applying BAB (0.001–350 µM) were normalized to drug-free controls and plotted versus the concentration. The smooth curve is a fit to a single-site model [I/Io = ((1 – c)/(1 + ([BAB]/KD)) + c] with a KD of 58.9 ± 11 nM and steady-state inhibition at high concentrations of 35 ± 0.5% (n = 9).

We therefore further investigated the mechanism of BAB inhibition using heterologously expressed Kv4 channels. BAB (0.04–40 µM) reduced the amplitude of the Kv4.2 current in a concentration-dependent manner (Fig. 5C). The smooth curve is a fit to a single-site model with a K_D of 59 ± 11 nM and steady-state inhibition of 35 ± 0.5% (n = 9). Despite applying high concentrations, BAB only partially inhibited the current, suggesting that Kv4.2 channels may not be completely blocked when the binding site is fully occupied. The BAB (1 µM) inhibition of Kv4.3 (34.2 ± 4.0%; n = 4) is not significantly different from that of Kv4.2 (30.7 ± 0.04%; n = 5) suggesting that the high-affinity inhibition may be similar for members of the Kv4 family.

We also examined the effects of BAB on Kv4.2 gating (Fig. 6). BAB (1 µM) caused a slight (–3 mV) hyperpolarizing shift in the midpoint of steady-state inactivation but did not alter the threshold of activation or the recovery from inactivation (Fig. 6, A and B). The time course of the current decay at +20 mV was fitted with the sum of two exponentials with time constants of 11.6 ± 1.0 and 66.0 ± 3.4 ms (n = 5), which was only slightly different from that of drug-free controls (_f = 13.4 ms, _s = 82.8 ms). Despite reducing the current amplitude by 30%, BAB did not seem to substantially alter Kv4.2 gating.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Effects of BAB on Kv4.2 activation and recovery from inactivation. The current of heterologously expressed Kv4.2 channels was compared before (control) and after bath application of BAB (1 µM). A, current was elicited by depolarizing to voltages between –80 and +30 mV in 10-mV increments. The normalized conductance was calculated as described previously (Fig. 2) and plotted versus the voltage. The smooth curves are fits to a fourth-order Boltzmann function with Vm and k values of –39.2 ± 2.3 and 20.7 ± 1.3 mV for controls (n = 5) and –40.3 ± 1.6 and 21.1 ± 0.9 mV after application of BAB (n = 3). Also plotted is the steady-state inactivation measured by applying 900-ms conditioning pulses to voltages between –95 and –40 mV before assessing the availability using a standard test pulse (20 mV/200 ms). The currents were normalized to those measured at –95 mV and plotted versus the test potential. The smooth curve is a fit to a Boltzmann function with a midpoint and slope factor of –63.4 ± 0.1 and 4.6 ± 0.1 mV for controls (n = 5) and –67.2 ± 0.1 and 5.1 ± 0.1 mV after BAB (n = 5). B, recovery was measured by depolarizing to +10 mV for 400 ms before returning to –100 mV for a variable interval (1–600 ms). Test pulses (10 mV/200 ms) applied at the end of the recovery interval were used to assess the recovery time course. The smooth curves are fits to an exponential function with recovery time constants of 84.9 ± 3.6 ms for control (n = 5) and 76.2 ± 2.6 ms after application of BAB (n = 4).

The S6 Segment of Kv4.2 Contributes to BAB Binding. The S6 membrane-spanning segment of voltage-gated K⁺ channels forms the cytoplasmic entrance of the channel and includes amino acids important for the binding of pore blockers (Aiyar et al., 1994; Lopez et al., 1994; Shieh and Kirsch, 1994; Yeola et al., 1996; Decher et al., 2004). Figure 7A shows that the primary amino acid sequence of the S6 segment is highly conserved among members of the Kv4 subfamily. Also shown is the helical wheel projection of the S6 segment of Kv4.2, indicating that several conserved hydrophobic amino acids (V397, L400, and V404) are aligned along the same face of the proposed -helix (Fig. 7B). V397 and L400 are located near the middle of the S6 segment just proximal to the conserved PVP motif, which is thought to induce a bend in the S6 -helix (Holmgren et al., 1998; Del Camino et al., 2000). V397, L400, and V404 are thought to line the cytoplasmic entrance of the Kv4 pore and are therefore well positioned to contribute to BAB binding.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Sequence alignment and proposed -helical structure of the S6 segment of Kv4 channels. A, aligned amino acid sequences of the S6 segments of the Kv4.1–Kv4.3 subunits. Boxes identify highly conserved residues believed to be located near the narrow region of the cytoplasmic pore. B, helical wheel projection of the Kv4.2 S6 segment showing that V397, L400, and V404 are predicted to be aligned along a common face of an -helix.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Effects of S6 mutations on the BAB inhibition of Kv4.2. A to C, V397T, L400T, and V404T mutants were expressed in tsA201 cells and currents elicited by depolarizing pulses (+20 mV/200 ms) from a holding potential of –100 mV. The currents are shown before (control) and after bath application of BAB (0.1–100 µM). D, peak currents of the S6 mutants measured in the presence of BAB were normalized to drug-free controls and plotted versus the concentration. The smooth curves are fits to a single-site model with parameters listed in Table 2. The dotted line is the fitted curve for the wild-type Kv4.2 replotted from Fig. 5C.

	Discussion

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BAB is a long-lasting anesthetic that is thought to act by inhibiting ion channels of DRG nociceptors. In this study, we examined the effects of BAB on the rapidly inactivating A-type K⁺ current (I_A) of acutely dissociated rat DRG neurons. We restricted our studies to small DRG neurons (<20 µm), which are thought to represent the cell bodies of unmyelinated nerve fibers (Harper and Lawson, 1985). These neurons express a variable component of A-type K⁺ current that was found to be insensitive to TEA (20 mM) but blocked by 4-AP (5 mM). The I_A component activated at voltages near the resting membrane potential of DRG neurons (–55 mV) and displayed rapid inactivation (Fig. 2). The pharmacology and gating properties of the DRG I_A were found to be similar to those of heterologously expressed Kv4 channels (Table 1). RT-PCR was used to establish that the messages encoding for the Kv4 channel subunits (Kv4.1–Kv4.3) are present in the rat DRG (Fig. 4). Direct comparison of the gating properties indicated that the native I_A component activated at more depolarized voltages and recovered from inactivation slowly by comparison to Kv4 channels. We think that some of these differences may be related to the overlap of the A-type current with slowly inactivating and sustained components of K⁺ current expressed in these neurons (Beekwilder et al., 2003). In addition, Kv4 channels are regulated by several auxiliary proteins (An et al., 2000; Nadal et al., 2001, 2003) that may be present in the native DRG neurons but absent in our heterologous expression system. Despite these differences, our data support the conclusion that Kv4 channels, either as homo- or heteromultimers, contribute to the rapidly inactivating A-type K⁺ current expressed in small DRG neurons. This conclusion is in good agreement with recent studies of DRG A-type K⁺ current (Fedulova et al., 1998; Sculptoreanu et al., 2004).

BAB was found to be a potent inhibitor of both the DRG I_A and Kv4.2 current (Fig. 5). Unfortunately, a detailed analysis of the properties of the BAB inhibition of the native current was complicated by the variable expression of I_A in small DRG neurons and difficulties isolating this component from the sustained K⁺ current expressed in these cells. Heterologously expressed Kv4 channels were therefore used to further investigate the mechanism of BAB inhibition. Kv4.2 channels were inhibited by BAB in a concentration-dependent manner, reaching a maximal inhibition of 35 ± 0.5% at concentrations >1 µM (Fig. 5C). Increasing the BAB concentration up to 350 µM did not further inhibit Kv4.2 consistent with a single high-affinity binding site with a K_D of 59 nM. BAB did not significantly alter the kinetics or voltage sensitivity of the current, indicating that the drug does not substantially affect Kv4.2 gating.

To further investigate the mechanism of BAB inhibition, we mutated amino acids of the membrane-spanning S6 segment of Kv4.2, which is known to form the cytoplasmic entrance of K⁺ channels and to contribute to the binding of pore blocking drugs (Aiyar et al., 1994; Lopez et al., 1994; Shieh and Kirsch, 1994; Yeola et al., 1996; Franqueza et al., 1997; Jerng et al., 1999; Caballero et al., 2003; Decher et al., 2004). V397, L400, and V404 are highly conserved in Kv4 channels and are aligned along a common face of the S6 -helix (Fig. 7). Residues equivalent to L400 in Kv2.1 and Kv3.1 channels contribute to the binding of internally applied TEA (Aiyar et al., 1994; Shieh and Kirsch, 1994). In Shaker and Kv1.5 channels, the threonine at position 397 is an important determinant of alkyl-TEA and quinidine binding (Choi et al., 1993; Yeola et al., 1996). Mutations at position 404 (V404I) weaken the local anesthetic and 4-aminopyridine block of Kv4 channels (Jerng et al., 1999; Caballero et al., 2003).

Substituting polar threonine at positions 397 and 400 significantly weakened (V397T) or nearly abolished (L400T) the BAB inhibition of the channel (Fig. 8D). The V397T and L400T mutations did not substantially alter Kv4.2 gating (Table 1), suggesting that threonine substitutions at these positions are well tolerated and do not indirectly alter BAB binding through changes in Kv4.2 gating. BAB is a relatively hydrophobic drug and at physiological pH is uncharged (pK_a = 2.6). Replacing hydrophobic valine and leucine residues of the S6 with the more polar threonine significantly increased the K_D, suggesting that hydrophobic interactions may contribute to BAB binding.

V397 and L400 are situated just proximal to the highly conserved PVP motif of Kv channels (Fig. 7A), which disrupts hydrogen bonding and induces bends in the S6 -helix (Del Camino et al., 2000; Del Camino and Yellen, 2001). In Shaker, residues positioned upstream of the S6 bend do not seem to participate in the conformational changes linked to channel opening (Hackos et al., 2002; Webster et al., 2004), which is consistent with our data showing that the V397T and L400T mutations do not alter Kv4.2 gating (Table 1). The specific effects of the V393T and L400T mutations on the K_D of BAB binding therefore supports the conclusion that these residues, which are proposed to be situated near the narrow region of the pore, are important determinants of drug binding. Our data indicate that BAB shares an overlapping binding site with drugs that inhibit K⁺ channels by pore blocking mechanisms (Choi et al., 1993; Aiyar et al., 1994; Jerng et al., 1999). However, our finding that BAB produced a partial inhibition of Kv4.2 current at high concentrations predicted to fully occupy the binding site suggests that the drug does not act by a simple pore-blocking mechanism, which predicts a complete inhibition of the current at high concentrations. Rather, when bound, BAB may not fully inhibit permeation through the channel, resulting in a reduction rather than a complete inhibition of K⁺ current. This mechanism is reminiscent of mutations near the cytoplasmic ends of the S6 segments of K⁺ channels that form the narrow region of the pore (Del Camino et al., 2000; Del Camino and Yellen, 2001; Zhou et al., 2001; Hackos et al., 2002). Inserting large aromatic amino acids within this region reduces the single-channel conductance, suggesting that the pore may narrow when bulky residues are substituted at these positions (Hackos et al., 2002). We speculate that molecular crowding caused by BAB binding near the narrow region of the pore may reduce, but not completely inhibit, K⁺ permeation through the channel. This would explain our finding that saturating concentrations BAB only partially inhibit Kv4.2 current.

By contrast, the PVP bend predicts that amino acids situated distal to the PVP motif (V404) may be situated within a wide vestibule rather than a narrow region of the pore (V397 and L400). This model is consistent with the observed effects of the V404T mutation that produced only a slight reduction in the affinity and steady-state BAB inhibition (Table 2) but induced a substantial hyperpolarizing shift in inactivation (Table 1). The data suggest that V404 may not play a direct role in BAB binding but that polar substitutions at this position may modify Kv4.2 gating or reduce the accessibility of the hydrophobic drug to its binding site situated near the narrow region of the pore.

Kv4 channels encode for a subthreshold-activating K⁺ current that inactivates at depolarized voltages and rapidly recovers at hyperpolarized potentials (Jerng et al., 2004). The A-type currents produced by these channels are important determinants of action potential duration (Bardoni and Belluzzi, 1993) and frequency of neuronal firing (Malin and Nerbonne, 2000). We speculate that the BAB inhibition of DRG Kv4 channels may slow repolarization after an action potential, thereby prolonging the refractory period and reducing the firing frequency of DRG neurons. In addition, our data suggest that Kv4 currents may contribute to the resting membrane potential of DRG neurons. The steady-state activation and inactivation of the DRG I_A component display considerable overlap between –40 and –60 mV (Fig. 2B), which is predicted to produce a persistent "window current" at voltages considered to be near the resting membrane potential of DRG neurons (–55 mV) (Christian et al., 1994; Sculptoreanu et al., 2004). A similar window current is also predicted for the heterologously expressed Kv4.2 channels (Fig. 3B) (Bahring et al., 2001). The BAB inhibition of Kv4-mediated window currents could lead to depolarization of the resting membrane potential. Although this might be expected to increase the excitability of DRG neurons, such changes in resting membrane potential could also indirectly modulate other voltage-gated ion channels expressed in nociceptors. For example, small DRG neurons express a unique combination of tetrodotoxin-sensitive and tetrodotoxin-resistant Na⁺ currents that inactivate between –100 and –30 mV (Wood et al., 2002). Depolarization of the resting membrane potential may reduce the steady-state availability of the Na⁺ channels that underlie the rapid upstroke of the DRG action potential (Van den Berg et al., 1995, 1996). We speculate that both the direct BAB inhibition of Kv4 and Kv1.1 channels (Beekwilder et al., 2003) and the enhanced state-dependent binding of BAB to voltage-gated Na⁺ channels (Van den Berg et al., 1996) may contribute to the long duration anesthesia produced by the epidural administration of BAB.

	Acknowledgements

 
We thank Drs. R. J. van den Berg, M. Covarrubias, and J. P. Beekwilder for helpful discussions and comments on the manuscript, and Dr. Jeanne Nerbonne for providing the cDNA for the Kv4.2 and Kv4.3 channels. We thank Susan Dunn for assistance with the RT-PCR studies.

	Footnotes

 
This work was supported by National Institute of General Medicine Grant GM5808.

doi:10.1124/jpet.105.087759.

ABBREVIATIONS: BAB, butamben, n-butyl-p-aminobenzoate; DRG, dorsal root ganglion; 4-AP, 4-aminopyridine; TEA, tetraethylammonium; RT-PCR, reverse transcription-polymerase chain reaction; PVP, proline 441/valine 442/proline 443.

Address correspondence to: Dr. Michael E. O'Leary, Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, 1020 Locust St., JAH 266, Philadelphia, PA 19107. E-mail: michael.oleary{at}jefferson.edu

	References

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