Stichodactyla helianthus Peptide, a Pharmacological Tool for Studying Kv3.2 Channels

    Department of Ion Channels, Merck Research Laboratories, Rahway, New Jersey (L.Y., J.H., P.M.D., R.M.Bu., R.S.S., P.B., R.M.Br., B.T.P., G.J.K., M.L.G.)
    Department of Physiology and Neuroscience, New York University School of Medicine, New York, New York (E.G., B.R.)

    Abstract

    Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well-characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. Chinese hamster ovary (CHO)-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. Stichodactyla helianthus peptide (ShK), isolated from S. helianthus venom and a known high-affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit 86Rb+ efflux from CHO-K1.hKv3.2b (IC50  0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus laevis oocytes or in planar patch-clamp studies, ShK inhibited hKv3.2b channels with IC50 values of 0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic  cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with the blockade of Kv3 channels (i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential after hyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections). Taken together, these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations.

    Voltage-gated potassium channels (Kv) represent a large family of proteins that regulate numerous physiological functions. Kv channels are tetrameric structures formed by the association of identical or closely related subunits. The study of potassium channels has been greatly facilitated by the discovery of high-affinity and selective channel inhibitors in the venoms of different organisms (Garcia et al., 1998; Corzo and Escoubas, 2003; Lewis and Garcia, 2003). Such peptidyl inhibitors have provided invaluable pharmacological tools for the isolation, purification, tissue localization, and the study of the structure and gating mechanism of ion channels (Garcia-Calvo et al., 1994).

    To date, many subtype-selective Kv channel inhibitors have been identified. Dendrotoxins (DTXs) from the venom of Dendroaspis angusticeps and BgK from the sea anemone Bunodosoma granuliferaare are potent blockers of Kv1.1, 1.2, and 1.6 channels (Harvey, 2001). Charybdotoxin (ChTX), a minor component of Leuiurus quinquestriatus venom, is a potent blocker of Kv1.3 and Kv1.2 channels (Castaneda et al., 1995; Garcia et al., 1999), whereas ShK, a peptide isolated from the sea anemone Stichodactyla helianthus, inhibits Kv1.1 and 1.3 channels with picomolar affinities (Middleton et al., 2003). Blood-depressing substances I and II from the venom of sea anemone Anemonia sulcata block Kv3.4 channels specifically (Diochot et al., 1998). Finally, phrixotoxins I and II from venom of the tarantula Phriotrichus auratus and hanatoxin from the venom of the spider Grammostola spatulata represent a class of gating-modifying peptides with activity against Kv4.2 and 4.3 (Diochot et al., 1999) and Kv2.1 channels (Swartz and MacKinnon, 1995), respectively.

    Kv3.2 is a member of the Kv3 channel subfamily. These channels possess rather unique fast activation at voltages positive to eC20 mV and very fast deactivation rates (Coetzee et al., 1999). Such biophysical properties could enable cells to fire high-frequency trains of action potentials, and therefore Kv3.2 could play a role in neuronal excitability. Consistent with this proposal, Kv3.2 is expressed in neurons that are known to fire at a high frequency, such as the inhibitory cortical GABAergic interneurons (Chow et al., 1999; Hernandez-Pineda et al., 1999; Tansey et al., 2002). In addition, Kv3.2 is expressed in the insulin-secreting, pancreatic  cells (Yan et al., 2004) and could contribute to the delayed-rectifier current in these cells. The  cell delayed-rectifier current is believed to regulate glucose-dependent firing, making it an attractive candidate for the development of novel glucose-dependent insulin secretagogues that could have usefulness in the treatment of type-2 diabetes. Although Kv3.2 channels are sensitive to low concentrations of tetraethylammonium ion (TEA) (Hernandez-Pineda et al., 1999), potent and selective Kv3.2 inhibitors with which to study the role of these channels in native tissues are not currently available.

    To search for Kv3.2 inhibitors, we generated CHO-K1 cells stably expressing the human Kv3.2b channel (CHO-K1.hKv3.2b cells) and established a functional 86Rb+ efflux assay with which to monitor the activity of the channel. Using this assay, ShK was identified as a potent inhibitor (IC50 = 0.59 nM) of heterologously expressed hKv3.2b channels and was used to evaluate the role of the channel in pancreatic  cells and in cortical GABAergic fast-spiking interneurons. ShK may serve as a useful pharmacological tool for studying Kv3.2 channels in native tissues.

    Materials and Methods

    Reagents. CHO-K1 cells stably expressing human Kv2.1 channels (CHO.K1.hKv2.1 cells) were obtained from Dr. O. Pongs (Institut fuer Neurale Signalverarbeitung, Hamburg, Germany). Rabbit polyclonal antibodies directed against the peptide (TPDLIGGDPGDDEDLAAKR) corresponding to residues 171 to 189 of human Kv3.2b were obtained as described previously (Chow et al., 1999). 86RbCl was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). ShK, ShK-lysine22-diaminopropionic acid (ShK-Dap22), apamin, and iberiotoxin were purchased from Peptides International Inc. (Louisville, KY). Kaliotoxin, agitoxin-I, agitoxin-II, and margatoxin were prepared as described previously (Suarez-Kurtz et al., 1999). TEA was purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were obtained from commercial sources and were of the highest purity available.

    Preparation of CHO-K1 Cells Stably Expressing hKv3.2b (CHO-K1.hKv3.2b Cells). CHO-K1 cells were maintained at 37°C in Ham's F-12 medium (Cellgro, Mediatech, VA) supplemented with 10% fetal bovine serum and appropriate antibiotics. cDNA coding for hKv3.2b (GenBank accession number AF268896 ), a splicing variant of hKv3.2 (Haas et al., 1993) was subcloned into pCI-neo mammalian expression vector (Promega, Madison, WI), and CHO-K1 cells were transfected using PolyFect Transfection Kit (QIAGEN, Valencia, CA), according to the manufacturer's instructions. Transfected cells were subjected to selection in the presence of 1 mg/ml G418. G418-resistant clones stably expressing functional hKv3.2b (CHO-K1.hKV3.2b cells) were identified by Western blot immunostaining and patch-clamp electrophysiological recordings.

    Western Blot Analysis. CHO-K1 or CHO-K1.hKv3.2b cells were rinsed with ice-cold phosphate-buffered saline and lysed in ice-cold lysis buffer containing 20 mM HEPES, pH 7.4, 1% Triton X-100, 150 mM NaCl, and 1x concentration of a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The cell lysates were cleared by centrifugation. Protein concentrations were determined using Bradford reagent (Bio-Rad, Hercules, CA). Cell lysates were resuspended in SDS loading buffer (Invitrogen, Carlsbad, CA) and proteins were separated using precast 4 to 20% gradient NuPAGE SDS-polyacrylamide gel electrophoresis gels (Invitrogen) and transferred to polyvinylidene difluoride membrane (Immobilon; Millipore Corporation, Billerica, MA). The membrane blot was first incubated with blocking buffer (5% nonfat dried milk, 2.5% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS in 1x Tris-buffered saline) overnight and then with blocking buffer containing rabbit hKv3.2 antiserum (1:500 dilution) for 2 h at room temperature. The blot was washed three times with blocking buffer, for 15 min each, and then incubated for 1 h in the same buffer with alkaline phosphatase-conjugated anti-rabbit secondary antibody. An enhanced chemifluorescence Western blotting kit (Amersham Biosciences, Piscataway, NJ) was used for detection. The blot was scanned using a Storm gel and blot imaging system (Amersham Biosciences).

    Conventional Patch-Clamp Electrophysiology. Membrane currents were recorded using standard whole-cell voltage-clamp techniques (Hamill et al., 1981) with a Dagan 3900A amplifier (Dagan, Minneapolis, MN). Microelectrodes fabricated from borosilicate glass were coated with Sylgard (Dow Corning, Midland, MI) and fire-polished. Electrode resistances were generally 2 to 4 M when filled with standard internal saline. The reference electrode was a silver-silver chloride wire within an agar bridge (4% agar in 170 mM KCl). Voltages given in the figures have not been corrected for the liquid junction potential between the internal and external solutions. All experiments were performed at room temperature (22eC25°C). Generation of analog voltage commands and digitization of membrane currents were controlled with PULSE software (HEKA Elektronik, Lambrecht, Germany) and an ITC-16 computer interface (InstruTECH Corporation, Port Washington, NY). Currents were digitized at 5 kHz and digitally filtered at 2 kHz. Digital subtraction of leakage and capacitive currents was performed by the P/n procedure, where n = 5. Voltage steps were applied every 5 to 30 s from a holding potential of eC80 mV.

    The standard pipette solution contained 130 mM potassium aspartate, 5 mM NaCl, 10 mM EGTA, 2 mM MgCl2, and 10 mM HEPES, with pH adjusted to 7.2 with 22 mM KOH. The standard external solution consisted of 160 mM NaCl, 1 mM KCl, 2.7 mM CaCl2, 0.5 mM MgCl2, and 5 mM HEPES, pH 7.5, with N-methyl-D-glucamine. For recordings of human islet cells, the pipette solution was 120 mM KCl, 20 mM KF, 10 mM EGTA, 10 mM HEPES, and 2 mM MgATP, with pH adjusted to 7.2 with KOH, and external solution was 160 mM NaCl, 4.5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 3 mM glucose, pH 7.2, with NaOH. Solutions were applied to cells by bath perfusion via gravity. The experimental chamber had a volume of 0.2 ml, and the perfusion rate was 1 to 2 ml/min. Flow of solution through the chamber was maintained at all times. TEA was added directly to the standard extracellular solution from a 1 M stock in water. Measurement of current amplitudes was performed with PULSEFIT software (version 8.53) (HEKA Elektronik). Fitting of current waveforms or extracted data was performed with PULSEFIT or Igor Pro 4.0 (WaveMetrics, Lake Oswego, OR). Results are reported as mean ± S.E.M.

    86Rb+ Efflux Assay. CHO-K1 hKv3.2b cells were plated into 96-well cell culture plates at a density of 2 x 105 cells/well and incubated with 6 e藽i/ml 86RbCl in 100 e of culture medium overnight at 37°C. To each well, 100 e of low-potassium buffer (135 mM NaCl, 4.6 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 0.2% bovine serum albumin, and 10 mM HEPES, pH 7.4, with NaOH) with or without test samples was added, and incubation continued for 30 min at 37°C. At the end of the incubation, the medium was replaced with 200 e of high-potassium buffer (140 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 0.2% bovine serum albumin, and 10 mM HEPES, pH 7.4, with KOH), with or without test samples, and 86Rb+ efflux was monitored for determined periods of time. To quantify the amount of 86Rb+ efflux, medium was removed from the wells, and 100 e was mixed with 100 e of MicroScint-20 (PerkinElmer Life and Analytical Sciences). 86Rb+ content of the cells was determined by the addition of 100 e of MicroScint-20. Efflux was defined as the percentage of 86Rb+ in each well in the low- or high-potassium buffer, normalized to the total radioactivity of the efflux solution and cells. IC50 values represent the mean ± S.E.M.

    Expression and Two-Electrode Voltage-Clamp Studies of hKv3.2 in X. laevis oocytes. X. laevis oocytes were prepared and injected using standard methods described previously (Arena et al., 1991). Adult female X. laevis oocytes were anesthetized with 0.17% tricaine methanesulfonate, and the ovaries were surgically removed and placed in a solution consisting of 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 2.5 mM sodium pyruvate, 100,000 U/l penicillin G, and 1000 mg/l streptomycin sulfate, pH 7.5 (modified OR-2). Ovarian lobes were broken open, rinsed several times in modified OR-2, and incubated in 0.2% collagenase (type 1; Sigma-Aldrich) in modified OR-2 with gentle shaking at room temperature. After 1 h, the collagenase solution was renewed, and the oocytes were incubated for an additional 30 to 90 min until approximately 50% of the oocytes were released from the ovaries. Stage V and VI oocytes were selected and placed in media containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, 2.5 mM sodium pyruvate, 0.5 mM theophylline, and 50 mg/ml gentamicin, pH 7.5 (ND-96) for 16 to 24 h before injection. Oocytes were injected with 50 nl of RNA encoding hKv3.2b at a concentration of 0.2 mg/ml. Oocytes were incubated at 18°C for 1 to 6 days in ND-96 before recording.

    Recordings were made at room temperature in modified ND-96 consisting of 96 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 3.5 mM BaCl2, and 5 mM HEPES, pH 7.5. Oocytes were voltage-clamped using a Dagan CA1 two-microelectrode amplifier (Dagan Corporation) interfaced to a Macintosh 7100/80 computer (Apple Computer, Cupertino, CA). The current-passing electrode was filled with 0.7 M KCl and 1.7 M potassium citrate, and the voltage-recording electrode was filled with 1 M KCl. Throughout the experiment, oocytes were superfused with modified ND-96 (control solution) or with ND-96 containing test peptides at a rate of approximately 3 ml/min. Data were acquired at 5 kHz and filtered at 1.7 kHz using Pulse software (version 8.53) from HEKA Elektronik. All recordings were performed from a holding potential of eC80 mV. IC50 values represent the mean ± S.D.

    Planar Patch-Clamp Electrophysiology. Parallel patch-clamp electrophysiology was performed essentially as described previously (Kiss et al., 2003). In brief, CHO-K1.hKv3.2b cells or CHO-K1.hKv2.1 cells were removed from culture plates using Versene (Invitrogen), subjected to centrifugation, and resuspended in 4 ml of Dulbecco's phosphate-buffered saline (Invitrogen) containing 138 mM NaCl, 8.1 mM Na3PO4, 2.67 mM KCl, 1.47 mM K3PO4, 0.9 mM CaCl2, and 0.5 mM MgCl2, pH 7.4. The internal solution consisted of 100 mM potassium gluconate, 40 mM KCl, 3.2 mM MgCl2, 3 mM EGTA, and 5 mM HEPES, pH 7.4, with KOH, supplemented with 120 e/ml amphotericin B. Voltage protocols and the recording of membrane currents were performed using the IonWorks HT software/hardware system (Molecular Devices, Sunnyvale, CA). Currents were sampled at 2.5 kHz, and leakage subtraction was performed using a 10-mV step from the holding potential (eC80 mV) and assuming a linear leak conductance. No correction for liquid junction potentials was used. Wells with seal resistances less than 70 M or less than 0.1 nA of Kv current at the test potential (+40 mV) were excluded from analysis. The mean seal resistance of the remaining wells was 169 M. Current amplitudes were calculated with the IonWorks software, fraction block was calculated in Microsoft Excel (Microsoft, Redmond, CA), and fitting was performed with Sigma Plot (SPSS Inc., Chicago, IL).

    Human Islet Cells. Human pancreatic islets were obtained from Dr. Jonathan Lakey (University of Alberta, Surgical-Medical Research Institute, Edmonton, AB, Canada) and the JDRF Human Islet Distribution Program at the University of Alberta (Edmonton, AB, Canada). Islets were isolated as described previously (Shapiro et al., 2000) using procedures approved by the University of Alberta Ethics Committee. Upon receipt of the tissue, islets were hand-picked, dissociated into single cells using trypsin, resuspended in culture medium, and plated on poly-L-lysineeCcoated glass chips. Cells were used for electrophysiology within 72 h of plating.

    Slice Preparation and Electrophysiology. Mice (14 days old) were anesthetized via intraperitoneal injection of pentobarbital and decapitated. All animal procedures complied with National Institutes of Health guidelines for the ethical use of animals. The brain was rapidly removed to oxygenated, ice-cold artificial cerebrospinal fluid, and sagittal slices (300 e thick) were cut and incubated in a holding chamber at room temperature for at least 1 h before electrophysiological recording, at which point they were transferred to a submersion-type recording chamber. The artificial cerebrospinal fluid used for cutting, incubation, and recording contained 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 20 mM glucose, 2 mM CaCl2, 2 mM MgSO4, and 0.5 mM ascorbic acid and was continuously bubbled with 95% O2 and 5% CO2 to maintain a pH of 7.40. Whole-cell current clamp recordings were made using an AxoClamp 2B amplifier (Axon Instruments Inc., Union City, CA). Patch electrodes (3eC5 M) were filled with a solution containing 130 mM potassium gluconate, 10 mM HEPES, 0.5 mM EGTA, 4.0 mM magnesium ATP, and 0.3 sodium GTP, with pH to 7.4 with KOH. Voltage output was filtered at 10 kHz, and data were acquired with a sampling frequency of 20 kHz. Neocortical GABAergic fast-spiking interneurons (FS cells), identified via multipolar morphology, lack of a prominent apical dendrite, and characteristic response to depolarizing current injection (Connors and Gutnick, 1990), were recorded in layer V primary somatosensory ("barrel") cortex. Values represent the mean ± S.D.

    Results

    Stable Expression of hKv3.2b in CHO-K1 Cells. To establish a robust, functional assay for identifying hKv3.2 inhibitors, we stably expressed the splice variant, hKv3.2b, in CHO-K1 cells. CHO-K1 cells were chosen because it is known that these cells express low levels of endogenous potassium channels, and they are routinely used for functional heterologous expression of Kv channels. Figure 1A shows a Western blot analysis of the CHO-K1.hKv3.2b cell clone that was selected for all subsequent studies. Two bands with estimated molecular masses of 57.5 and 74 kDa are specifically recognized by the Kv3.2 antibody in the lysate of these cells (lane 2) but not in the lysate of the parental, untransfected CHO-K1 cells (lane 1). The upper band agrees well with the predicted molecular mass of the full-length protein, whereas the lower band probably represents a proteolytic product of the higher molecular mass product.

    Electrophysiological Properties of CHO-K1.hKv3.2b Cells. The biophysical properties of heterologously expressed hKv3.2b channels were evaluated in patch-clamp recordings. Characteristics of the Kv3 channel subfamily (including Kv3.2) include activation at potentials more positive than approximately eC20 mV and fast deactivation upon repolarization (Rudy and McBain, 2001). Whereas the parental untransfected CHO-K1 cells display little if any Kv current (data not shown), CHO-K1.hKv3.2b cells displayed large outward currents in response to step depolarizations (Fig. 1B). The currents activated rapidly ( = 3.0 ± 0.9 ms at +40 mV, n = 5) and deactivated rapidly ( = 5.6 ± 0.2 ms at eC40 mV, n = 5). The voltage dependence of activation was studied by measuring the amplitude of tail currents after step depolarizations to various potentials. For the cell shown in Fig. 1B, the voltage-dependence of activation was well-described by a Boltzmann distribution with a V50 of +22.6 mV and a slope of 13.5 mV (Fig. 1C). On average, the V50 was +20.2 ± 0.9 mV and the slope was 14.2 ± 0.5 mV (n = 5). To study current inactivation, long depolarizations (7 s) to various potentials were given (Fig. 1D). Currents decayed slowly at all potentials tested. At +40 mV, the current decayed 28 ± 7% (n = 3) over the 7-s step. Kv3 currents are known to be sensitive to low concentrations of TEA (Hernandez-Pineda et al., 1999), and this was also true for the channels present in CHO-K1.hKv3.2b cells (Fig. 1E). In these experiments, TEA reversibly inhibited hKv3.2b currents in a dose-dependent fashion. The combined data from experiments carried out in four cells illustrate that TEA blocked hKv3.2b with an IC50 of 0.3 mM (Fig. 1F). Taken together, the data indicate that CHO-K1.hKv3.2b cells display a TEA-sensitive current with characteristics consistent with Kv3.2.

    TEA-Sensitive 86Rb+ Efflux from CHO-K1.hKv3.2b Cells. A functional, high-capacity assay for identifying hKv3.2 inhibitors was developed on the basis of the ability of 86Rb+ to permeate through potassium channels. Cells incubated with 86RbCl accumulate 86Rb+ through operation of the Na/K-ATPase pump. When CHO-K1.hKv3.2b cells were then exposed to physiological, low external potassium (low-K) conditions, a time-dependent efflux of 86Rb+ from the cells occurred. In the presence of high external potassium, however, 86Rb+ efflux occurred much more rapidly, reflecting the activity of Kv channels. The Kv channel-mediated component of 86Rb+ efflux, defined as the difference between efflux under high-K and low-K conditions, reached a plateau in approximately 10 min (Fig. 2A). 86Rb+ efflux from CHO-K1.hKv3.2b cells was inhibited by TEA in a dose-dependent manner with an IC50 of 0.25 ± 0.01 mM (n = 3) (Fig. 2B). High-K dependent 86Rb+ efflux was not observed in the parental, untransfected CHO-K1 cells, although the basal levels of efflux in low-K were similar to those of CHO-K1.hKv3.2b cells (data not shown). These data suggest that in CHO-K1.hKv3.2b cells, high-KeCinduced 86Rb+ efflux reflects the activity of hKv3.2 channels. The high-capacity, 86Rb+ functional assay provides a means for identifying Kv3.2 inhibitors.

    ShK Is a Potent Inhibitor of hKv3.2b Channels. Using the 86Rb+ efflux assay described above, we evaluated a number of scorpion venoms and several known potassium channel inhibitors for their ability to block hKv3.2b channels. All venoms and the majority of potassium channel inhibitors such as apamin, kaliotoxin, iberiotoxin, agitoxin-I, agitoxin-II, margatoxin, and ChTX, when tested at concentrations up to 300 nM, did not significantly inhibit 86Rb+ efflux from the CHO-K1.hKv3.2b cells (data not shown). In marked contrast, 100 nM ShK caused complete inhibition of 86Rb+ efflux, and ShK-Dap22, in which diaminopropionic acid (Dap) was substituted at lysine 22 of ShK (Kalman et al., 1998), also inhibited 86Rb+ efflux to a lesser extent. Dose-response curves for inhibition of 86Rb+ efflux from CHO-K1.hKv3.2b cells indicate that ShK and ShK-Dap-22 block hKv3.2b-mediated 86Rb+ efflux with IC50 values of 0.62 ± 0.10 (n = 3) and 83.2 ± 22.1 (n = 3) nM, respectively, and Hill coefficients of 1 (Fig. 2C). These results provide the first evidence that ShK is a potent inhibitor of hKv3.2b channels.

    ShK Inhibits hKv3.2b but Not hKv2.1 Channels. Because both Kv2.1 and Kv3.2 channels are expressed in human pancreatic  cells and either one or both channels could contribute to the delayed-rectifier potassium current that regulates the firing frequency of the cells in the presence of high glucose, we compared the ability of ShK to inhibit both channels. X. laevis oocytes expressing hKv3.2b exhibited rapidly activating currents in response to step depolarizations to +20 and +80 mV (Fig. 3A) that were not seen in uninjected or water-injected oocytes. Similar to hKv3.2b currents expressed in mammalian cell lines, the hKv3.2b current in oocytes was blocked by TEA with an IC50 of 0.10 ± 0.01 mM (n = 3) (data not shown). Bath application of 0.3 nM ShK reduced the current activated by depolarizations to +20 and +80 mV (Fig. 3A, gray line). This inhibition of current amplitude was concentration-dependent, shown in Fig. 3B for the current at +80 mV, and displayed an IC50 value of 0.31 ± 0.06 nM (n = 6).

    In an automated patch-clamp system (IonWorks HT) that performs synchronous perforated patch recordings in a 384-wells planar array (Schroeder et al., 2003), the blocking potency of ShK against hKv3.2b or Kv2.1 channels was evaluated after a 10-min incubation period in either peptide or vehicle. ShK inhibited hKv3.2b current in a dose-dependent manner with an IC50 value of 6 nM but had no effect on hKv2.1 current at concentrations up to 1 e (Fig. 3D). These results suggest that ShK can be used as a pharmacological tool to evaluate the contribution of Kv3.2 channels to the whole-cell currents of cells that express both Kv3.2 and Kv2.1 channels. One such cell type is the human pancreatic  cell.

    ShK Does Not Inhibit the Delayed Rectifier Current of Human  Cells. Whole-cell voltage-clamp recordings from human  cells, identified by the presence of insulin mRNA through single-cell reverse-transcriptase polymerase chain reaction, show a prominent delayed-rectifier current with a voltage-dependence consistent with Kv2 and Kv3 channels (J. Herrington, M. Sanchez, D. Wunderler, L. Yan, R. M. Bugianesi, I. E. Dick, S. A. Clark, R. M. Brochu, B. T. Priest, M. G. Kohler, and O. B. McManus, submitted manuscript). Application of 100 nM ShK did not significantly inhibit (7 ± 4%, n = 12) this current (Fig. 4, A and B). TEA is known to inhibit the delayed-rectifier current of rodent  cells and enhance glucose-dependent insulin secretion (MacDonald and Wheeler, 2003). Likewise, the delayed-rectifier current of human  cells was blocked by TEA (Fig. 4, C and D). Application of 1 and 10 mM TEA inhibited the delayed rectifier current by 51 ± 2% (n = 20) and 89 ± 2%%(n = 19), respectively. Thus, the delayed rectifier of human  cells is TEA-sensitive and ShK-insensitive.

    The Effects of ShK on Fast-Spiking Cortical Interneurons Are Consistent with Block of Kv3.2 Channels. The presence of Kv3 channels in cortical GABAergic FS cells is required for the rapid repolarization of action potentials as well as for the initiation and maintenance of high-frequency firing. Application of low concentrations of TEA (a nonselective blocker of Kv3 channels) or genetic elimination of Kv3 channels produces broadening of the FS cell action potential, ablation of the afterhyperpolarization (AHP), and an attenuation of maximal achievable firing frequencies by these cells (Erisir et al., 1999; Lau et al., 2000; Rudy and McBain, 2001). Bath application of 100 nM ShK had no effect on resting membrane potential (Vm; eC73.0 ± 5.5 and eC75.0 ± 5.2 mV in control and after addition of ShK, respectively; n = 4) but produced a small increase in membrane resistance (Rm) from 112 ± 52 M under control conditions to 139 ± 61 M after ShK application (an increase of 26 ± 20% over control; n = 4). However, consistent with blockade of Kv3 channels, bath application of 100 nM ShK produced an increase in action potential half-width (i.e., the width of the action potential at half-amplitude, measured from action potential threshold to action potential peak) from 0.41 ± 0.05 to 0.58 ± 0.16 ms (n = 4), a decrease in the amplitude of the AHP as measured from action potential threshold to the maximal negativity of the afterhyperpolarization from 19.9 ± 8.1 to 14.1 ± 8.0 mV (n = 4), and a decrease in maximum firing frequency in response to depolarizing current injections from 280 ± 53 to 181 ± 93 Hz (n = 4) (Fig. 5). Statistical significance at the p < 0.05 level was achieved via unpaired, two-tailed t test for AHP and maximal firing frequency but not for the other parameters shown in Fig. 5F. In contrast, 100 nM dendrotoxin-I, a selective inhibitor of Kv channels containing Kv1.1, Kv1.2, and/or Kv1.6 subunits, has no effect on the FS cell action potential and, in fact, produces a slight increase in firing frequency (Erisir et al., 1999). However, ShK did produce a marked increase in the frequency of spontaneous postsynaptic potentials recorded from FS cells (data not shown). This effect is qualitatively similar to that produced by 100 nM dendrotoxin-I and is consistent with blockade of Kv1 channels, probably present in the presynaptic terminals of intracortical excitatory synapses (Coetzee et al., 1999; Bekkers and Delaney, 2001; Harvey, 2001).

    Discussion

    The goal of the present study was to identify potent and selective inhibitors of Kv3.2 channels with which to investigate the physiological role of the channel in native preparations. To this end, we constructed a CHO-K1.hKv3.2b cell line that stably expresses hKv3.2b channels and established a functional 86Rb+ efflux assay that was used to screen a variety of venoms and known potassium-channel inhibitors. ShK, a peptide isolated from S. helianthus venom, was found to inhibit hKv3.2b-mediated 86Rb+ efflux from CHO-K1.hKv3.2b cells in a dose-dependent manner and with high potency (IC50 = 0.62 nM). In electrophysiological recordings from X. laevis oocytes expressing hKv3.2b channels, ShK displayed an IC50 of 0.31 nM for channel inhibition, whereas in CHO-K1.hKv3.2b cells, the IC50 was 6 nM measured by planar patch-clamp electrophysiology. Taken together, these results suggest that ShK is a potent inhibitor of heterologously expressed hKv3.2b channels.

    ShK is a potent inhibitor of certain members of the Kv1-channel family. In particular, ShK blocks Kv1.1 and Kv1.3 channels with IC50 values of 1 pM, a 1000-fold lower concentration than that required to inhibit Kv3.2 channels. Evidence also suggests that native Kv currents in the central nervous system, which are predominantly carried by Kv1.2 channels, are highly sensitive to the peptide (Middleton et al., 2003). Thus, the usefulness of ShK in discerning the physiological role of Kv3.2 channels must be considered with caution in preparations in which other ShK-sensitive channels may be present. This is particularly relevant to the central nervous system, given the large diversity of Kv channels that exist in different neurons and the wide distribution of Kv1 subunits. Nonetheless, in cells in which Kv1 channels are present, other Kv1 specific peptides, such as DTX, could be used to distinguish between Kv3- and Kv1-mediated effects of ShK. In other systems, in which channel distribution is more restricted and better defined, ShK provides a pharmacological tool for studying Kv3.2 channels. For instance, polymerase chain reaction, in situ hybridization, and immunostaining with specific Kv antibodies have provided strong evidence for the presence of Kv2.1- and Kv3.2-channel subunits in pancreatic  cells (MacDonald et al., 2002; Yan et al., 2004). Because the association of Kv2.1 and Kv3.2 subunits has not been demonstrated either in vitro or in vivo, the delayed-rectifier Kv current in  cells is likely to be carried by channels that contain either Kv2.1 or Kv3.2 subunits. Defining the subunit composition of the delayed-rectifier current in  cells is important because this current regulates firing frequencies in the presence of glucose, and inhibitors of this current would be expected to enhance glucose-dependent insulin secretion and therefore have usefulness in the treatment of type-2 diabetes. ShK does not inhibit hKv2.1 currents, and because Kv1.1 subunits are not found in human pancreatic islets and functional Kv1.3 channels have not been reported in human  cells (MacDonald and Wheeler, 2003; Yan et al., 2004), the putative contribution of Kv3.2 subunits to the -cell Kv current can be evaluated. Even when high concentrations of ShK were used, no inhibition of the human delayed-rectifier current in  cells was observed. Although these experiments strongly suggest that a homomultimeric Kv3.2 channel is not a component of the  cell delayed-rectifier current, it raises the question of why the channel subunit is expressed in these cells. It is possible that coexpression of Kv3.2 with other ShK-insensitive subunits could lead to a tetrameric complex insensitive to the peptide, and additional experiments are needed to evaluate this possibility.

    Cortical GABAergic FS cells are named for their ability to fire sustained trains of brief action potentials at high frequency and, in fact, display the highest firing rates of any cortical cell (McCormick et al., 1985; Connors and Gutnick, 1990). Cortical FS cells express Kv3.1 and Kv3.2 subunits. A large body of evidence now exists to support the conclusion that the fast-spiking phenotype is, in fact, determined by the somatic expression of these Kv3 channels (Rudy and McBain, 2001; Lien and Jonas, 2003), which activate at depolarized potentials (relative to other Kv channels) and deactivate rapidly. Even at high firing frequencies, the effect of activation of Kv3.1/Kv3.2 channels is largely restricted to the repolarization phase of the action potential (Du et al., 1996), with little direct effect on the rate of rise or on spike threshold, and the interspike interval is established to a large degree by the accumulation of voltage-gated sodium-channel inactivation. However, by increasing the rate of spike repolarization (and keeping action potentials brief), Kv3.1/Kv3.2 channels limit sodium-channel inactivation. Moreover, the large, fast afterhyperpolarization elicited by Kv3.1/Kv3.2 currents accelerates recovery from inactivation of sodium channels (Rudy and McBain, 2001). Thus, Kv3.1/Kv3.2 channels ensure that sufficient sodium channels are available for a subsequent action potential, with little counteractive Kv current. The effects of pharmacological blockade or genetic elimination of Kv3.1/Kv3.2 channels has provided strong evidence that these channels are critical for high-frequency repetitive firing in FS cells (Martina et al., 1998; Erisir et al., 1999; Lau et al., 2000), as has a recent study using dynamic clamp technology (Lien and Jonas, 2003). In this context, the results of this study concerning the effect of ShK in FS cells are consistent with the proposed role for Kv3.2 channels and support the use of the peptide for studying these channels in other native preparations. Contribution of Kv1.1 and/or Kv1.3 channels to the observed effects of ShK in FS cells does not seem likely because DTX-I and ChTX, potent inhibitors of Kv1.1 and Kv1.3 channels, respectively, do not reproduce the effects of ShK on the shape of the FS cell action potential and on repetitive firing. In addition, Kv1.3 channels with their very slow recovery from inactivation would not be able to contribute to the fast firing frequency of FS cells.

    A significant difference in the inhibitory potency of ShK on Kv3.2b channels was observed between X. laevis oocyte electrophysiology (IC50 = 0.31 nM) and planar patch-clamp electrophysiology on CHO-K1.hKv3.2b cells (IC50 = 6 nM) (Fig. 3, C and D). Indeed, IC50 values for inhibition of 86Rb+ flux from CHO-K1.hKv3.2b cells are closer to those obtained from electrophysiological experiments on oocytes. Although the reasons for the block affinity of ShK in the planar patch-clamp system are not fully understood, several possibilities, such as an incomplete equilibration and adsorption to the planar array caused by the high surface-to-volume ratio, could account for the observed differences.

    In summary, we have provided evidence that ShK is a potent inhibitor of the hKv3.2b channel. Although much remains to be learned about the structural basis of the interaction between ShK and the Kv3.2 channel, it is likely that ShK will serve as an important pharmacological tool for the study of Kv3 family channels in native systems, particularly in those situations in which Kv1 channels are not present or can be selectively modulated.

    L.Y., J.H., and E.G. contributed equally to this work.

    doi:10.1124/mol.105.011064.

    References

    Arena JP, Liu KK, Paress PS, and Cully DF (1991) Avermectin-sensitive chloride currents induced by Caenorhabditis elegans RNA in Xenopus oocytes. Mol Pharmacol 40: 368eC374.

    Bekkers JM and Delaney AJ (2001) Modulation of excitability by alpha-dendrotoxin-sensitive potassium channels in neocortical pyramidal neurons. J Neurosci 21: 6553eC6560.

    Castaneda O, Sotolongo V, Amor AM, Stocklin R, Anderson AJ, Harvey AL, Engstrom A, Wernstedt C, and Karlsson E (1995) Characterization of a potassium channel toxin from the Caribbean Sea anemone Stichodactyla helianthus. Toxicon 33: 603eC613.

    Chow A, Erisir A, Farb C, Nadal MS, Ozaita A, Lau D, Welker E, and Rudy B (1999) K+ channel expression distinguishes subpopulations of parvalbumin- and somatostatin-containing neocortical interneurons. J Neurosci 19: 9332eC9345.

    Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, et al. (1999) Molecular diversity of K+ channels. Ann NY Acad Sci 868: 233eC285.

    Connors BW and Gutnick MJ (1990) Intrinsic firing patterns of diverse neocortical neurons. Trends Neurosci 13: 99eC104.

    Corzo G and Escoubas P (2003) Pharmacologically active spider peptide toxins. Cell Mol Life Sci 60: 2409eC2426.

    Diochot S, Drici MD, Moinier D, Fink M, and Lazdunski M (1999) Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol 126: 251eC263.

    Diochot S, Schweitz H, Beress L, and Lazdunski M (1998) Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4. J Biol Chem 273: 6744eC6749.

    Du J, Zhang L, Weiser M, Rudy B, and McBain CJ (1996) Developmental expression and functional characterization of the potassium-channel subunit Kv3.1b in parvalbumin-containing interneurons of the rat hippocampus. J Neurosci 16: 506eC518.

    Erisir A, Lau D, Rudy B, and Leonard CS (1999) Function of specific K+ channels in sustained high-frequency firing of fast-spiking neocortical interneurons. J Neurophysiol 82: 2476eC2489.

    Garcia ML, Hanner M, and Kaczorowski GJ (1998) Scorpion toxins: tools for studying K+ channels. Toxicon 36: 1641eC1650.

    Garcia ML, Hanner M, Knaus HG, Slaughter R, and Kaczorowski GJ (1999) Scorpion toxins as tools for studying potassium channels. Methods Enzymol 294: 624eC639.

    Garcia-Calvo M, Knaus HG, McManus OB, Giangiacomo KM, Kaczorowski GJ, and Garcia ML (1994) Purification and reconstitution of the high-conductance, calcium-activated potassium channel from tracheal smooth muscle. J Biol Chem 269: 676eC682.

    Haas M, Ward DC, Lee J, Roses AD, Clarke V, D'Eustachio P, Lau D, Vega-Saenz de Miera E, and Rudy B (1993) Localization of Shaw-related K+ channel genes on mouse and human chromosomes. Mamm Genome 4: 711eC715.

    Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflueg Arch Eur J Physiol 391: 85eC100.

    Harvey AL (2001) Twenty years of dendrotoxins. Toxicon 39: 15eC26.

    Hernandez-Pineda R, Chow A, Amarillo Y, Moreno H, Saganich M, Vega-Saenz de Miera EC, Hernandez-Cruz A, and Rudy B (1999) Kv3.1-Kv3.2 channels underlie a high-voltage-activating component of the delayed rectifier K+ current in projecting neurons from the globus pallidus. J Neurophysiol 82: 1512eC1528.

    Kalman K, Pennington MW, Lanigan MD, Nguyen A, Rauer H, Mahnir V, Paschetto K, Kem WR, Grissmer S, Gutman GA, et al. (1998) ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide. J Biol Chem 273: 32697eC32707.

    Kiss L, Bennett PB, Uebele VN, Koblan KS, Kane SA, Neagle B, and Schroeder K (2003) High throughput ion-channel pharmacology: planar-array-based voltage clamp. Assay Drug Dev Technol 1: 127eC135.

    Lau D, Vega-Saenz de Miera EC, Contreras D, Ozaita A, Harvey M, Chow A, Noebels JL, Paylor R, Morgan JI, Leonard CS, et al. (2000) Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins. J Neurosci 20: 9071eC9085.

    Lewis RJ and Garcia ML (2003) Therapeutic potential of venom peptides. Nat Rev Drug Discov 2: 790eC802.

    Lien CC and Jonas P (2003) Kv3 potassium conductance is necessary and kinetically optimized for high-frequency action potential generation in hippocampal interneurons. J Neurosci 23: 2058eC2068.

    MacDonald PE, Sewing S, Wang J, Joseph JW, Smukler SR, Sakellaropoulos G, Saleh MC, Chan CB, Tsushima RG, Salapatek AM, et al. (2002) Inhibition of Kv2.1 voltage-dependent K+ channels in pancreatic beta-cells enhances glucose-dependent insulin secretion. J Biol Chem 277: 44938eC44945.

    MacDonald PE and Wheeler MB (2003) Voltage-dependent K+ channels in pancreatic beta cells: role, regulation and potential as therapeutic targets. Diabetologia 46: 1046eC1062.

    Martina M, Schultz JH, Ehmke H, Monyer H, and Jonas P (1998) Functional and molecular differences between voltage-gated K+ channels of fast-spiking interneurons and pyramidal neurons of rat hippocampus. J Neurosci 18: 8111eC8125.

    McCormick DA, Connors BW, Lighthall JW, and Prince DA (1985) Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex. J Neurophysiol 54: 782eC806.

    Middleton RE, Sanchez M, Linde AR, Bugianesi RM, Dai G, Felix JP, Koprak SL, Staruch MJ, Bruguera M, Cox R, et al. (2003) Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile. Biochemistry 42: 13698eC13707.

    Rudy B and McBain CJ (2001) Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing. Trends Neurosci 24: 517eC526.

    Schroeder K, Neagle B, Trezise DJ, and Worley J (2003) Ionworks HT: a new high-throughput electrophysiology measurement platform. J Biomol Screen 8: 50eC64.

    Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, and Rajotte RV (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343: 230eC238.

    Suarez-Kurtz G, Vianna-Jorge R, Pereira BF, Garcia ML, and Kaczorowski GJ (1999) Peptidyl inhibitors of Shaker-type Kv1 channels elicit twitches in guinea pig ileum by blocking Kv1.1 at enteric nervous system and enhancing acetylcholine release. J Pharmacol Exp Ther 289: 1517eC1522.

    Swartz KJ and MacKinnon R (1995) An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. Neuron 15: 941eC949.

    Tansey EP, Chow A, Rudy B, and McBain CJ (2002) Developmental expression of potassium-channel subunit Kv3.2 within subpopulations of mouse hippocampal inhibitory interneurons. Hippocampus 12: 137eC148.

    Yan L, Figueroa DJ, Austin CP, Liu Y, Bugianesi RM, Slaughter RS, Kaczorowski GJ, and Kohler MG (2004) Expression of voltage-gated potassium channels in human and rhesus pancreatic islets. Diabetes 53: 597eC607.