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NEUROPHARMACOLOGY

Iptakalim Modulates ATP-Sensitive K⁺ Channels in Dopamine Neurons from Rat Substantia Nigra Pars Compacta

Division of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona (J.W., J.H., J.D., Q.L., K.-C.Y.); Department of Cardiovascular Pharmacology, Institute of Pharmacology and Toxicology, Beijing, People's Republic of China (J.W., Y.-P.C., H.W.); Department of Medical Technology, Hirosaki University School of Health Science, Hirosaki, Japan (T.T.); Department of Physiology I, Hirosaki University School of Medicine, Hirosaki, Japan (S.S., M.W.); Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona (P.A.S.); and Department of Pharmacology, Nanjing University of Medical Science, Nanjing, People's Republic of China (J.W., J.H., G.H.)

Received April 14, 2006; accepted July 10, 2006.

	Abstract

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Iptakalim, a novel cardiovascular ATP-sensitive K⁺ (K_ATP) channel opener, exerts neuroprotective effects on dopaminergic (DA) neurons against metabolic stress-induced neurotoxicity, but the mechanisms are largely unknown. Here, we examined the effects of iptakalim on functional K_ATP channels in the plasma membrane (pm) and mitochondrial membrane using patch-clamp and fluorescence-imaging techniques. In identified DA neurons acutely dissociated from rat substantia nigra pars compacta (SNc), both the mitochondrial metabolic inhibitor rotenone and the sulfonylurea receptor subtype (SUR) 1-selective K_ATP channel opener (KCO) diazoxide induced neuronal hyperpolarization and abolished action potential firing, but the SUR2B-selective KCO cromakalim exerted little effect, suggesting that functional K_ATP channels in rat SNc DA neurons are mainly composed of SUR1. Immunocytochemical staining showed a SUR1-rather than a SUR2B-positive reaction in most dissociated DA neurons. At concentrations between 3 and 300 µM, iptakalim failed to hyperpolarize DA neurons; however, 300 µM iptakalim increased neuronal firing. In addition, iptakalim restored DA neuronal firing during rotenone-induced hyperpolarization and suppressed rotenone-induced outward current, suggesting that high concentrations of iptakalim close neuronal K_ATP channels. Furthermore, in human embryonic kidney 293 cells, iptakalim (300-500 µM) closed diazoxide-induced Kir6.2/SUR1 K_ATP channels, which were heterologously expressed. In rhodamine-123-preloaded DA neurons, iptakalim neither depolarized mitochondrial membrane nor prevented rotenone-induced mitochondrial depolarization. These data indicate that iptakalim is not a K_ATP channel opener in rat SNc DA neurons; instead, iptakalim is a pm-K_ATP channel closer at high concentrations. These effects of iptakalim stimulate further pharmacological investigation and the development of possible therapeutic applications.

	Materials and Methods

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Acutely Dissociated DA Neurons from Rat SNc. Single DA neurons were acutely dissociated from 2- to 3-week-old Wistar rats using a previously described method (Wu and Partridge, 1998; Wu et al., 2002, 2004). In brief, each rat was anesthetized using halothane, and the brain was rapidly removed. Several 400-µm coronal slices, which contained the SNc, were cut using a vibratome (Vibroslice 725M; WPI, Sarasota, FL) in cold (2-4°C) artificial cerebrospinal fluid (ACSF) and continuously bubbled with carbogen (95% O₂-5% CO₂). The slices were then incubated in a preincubation chamber (Warner Ins., Holliston, MA) and allowed to recover for at least 1 h at room temperature (22 ± 1°C) in oxygenated ACSF. Thereafter, the slices were treated with pronase (1 mg/6 ml) at 31°C for 30 min and subsequently treated with the same concentration of thermolysin for another 30 min. The SNc was micropunched out from the slices using a well polished needle. Each punched piece was then dissociated mechanically using several fire-polished micro-Pasteur pipettes in a 35-mm culture dish filled with well oxygenated standard external solution (composition defined hereafter). The separated single cells usually adhered to the bottom of the dish within 30 min. In the present study, we used only those SNc neurons that maintained original morphological features of polygonal, large (20-30 µm), or medium (15-20 µm) somata with two to four thick, primary dendritic processes.

Immunocytochemical Staining to Identify Dissociated DA Neurons and SUR1 and SUR2B Protein Expression. For identification of DA neurons, single dissociated neurons were fixed for 5 min with methanol at -20°C. After fixation, the cells were permeabilized with 1 mg/ml saponin (Sigma Chemical Co., St. Louis, MO) in water for 5 min at room temperature. The samples were incubated overnight at 4°C with tyrosine hydroxylase (TH) primary antibody (AB152; Chemicon International Inc., Temecula, CA) in phosphate-buffered saline (PBS), which contained 5% bovine serum albumin. The cells were then rinsed with PBS and incubated with cy3-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. After three additional rinses with PBS, the samples were examined under a Zeiss fluorescence microscope (Zeiss, Oberkochen, Germany), and images were processed using Photoshop (Adobe Systems Inc., San Jose, CA; Fig. 1A). To identify protein expression of SUR1 and SUR2B subunits of K_ATP channels, single dissociated SNc neurons were fixed and permeabilized in the same manner as described previously. The samples were then incubated overnight in SUR1 or SUR2B primary antibody (sc-25683 and sc-5793, respectively; Santa Cruz Biotechnology Inc., Santa Cruz, CA). The cells were subsequently rinsed with PBS and incubated with fluorescein isothiocyanate-conjugated secondary antibodies (sc-2012 and sc-2024; Santa Cruz Biotechnology Inc.) for at least 2 h before final rinses and fluorescence microscopy. Both the primary and secondary antibodies were diluted with PBS, which contained 5% bovine serum albumin.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Functional KATP channels in dissociated SNc neurons. A, spontaneous action potential firing (3 Hz, Aa) was sensitive to dopamine. The duration (at a 50% level of amplitude) of single spikes was longer than 2 ms (Ab). B, hyperpolarizing activated conductance was demonstrated by voltage-clamp (Ba) and current-clamp (Bb) recordings. C, representative of a single dissociated neuron from the SNc. After perforated whole-cell recording (phase-contrast photo-picture, Ca), brief suction was applied to convert to whole-cell recording configuration, and Lucifer yellow (1 mg/ml in the recording pipette) was delivered to the recorded cell by a 5-mV hyperpolarizing pulse (0.5 Hz) for 3 min. The labeled neuron was identified using a fluorescence microscope (Cb) and showed a positive TH reaction (Cc). D, SUR1-selective KCO diazoxide hyperpolarized DA neurons and reduced action potential firing. E, SUR2B-selective KCO cromakalim neither hyperpolarized SNc DA neurons nor affected action potential firing. F, rotenone (100 nM) induced membrane hyperpolarization and eliminated action potential firing, and these effects were reversed by the KATP channel blocker tolbutamide. Horizontal dashed line in D to F, level of resting membrane potential.

Heterologous Expression of Kir6.2/SUR1 K_ATP Channels in Cultured HEK-293 Cells and Whole-Cell Patch-Clamp Recordings. HEK-293 cells were cultured in plastic dishes (9.4 cm in diameter) at 37°C in a humidified atmosphere of 95% air and 5% CO₂ in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. Cells were transiently transfected using Lipofectamine reagent (Lipofectamine 2000; Invitrogen) mixed with pcDNA3.1 vector, which contained cDNA encoding SUR1 or Kir6.2 in serum-free medium. As a control, cells were transfected with pcDNA3.1 vector alone. The cells were transiently cotransfected with vectors encoding SUR1 and Kir6.2 subunits at a molar plasmid ratio of 1:2. A marker gene encoding green fluorescent protein (pEGFP-N1; Clontech, Mountain View, CA) was cotransfected with the cDNA at a ratio of 1:10 to allow the transfected cells to be identified by epifluorescence. Transfection was performed according to the manufacturer's instructions. The cells were allowed to express the products of the transfected DNA for 48 h and were then used for electrophysiological experiments. Conventional whole-cell patch-clamp recordings were used to test the functional Kir6.2/SUR1 K_ATP channels expressed in cultured HEK-293 cells. Whole-cell currents were recorded in voltage-clamp mode. Series resistance during whole-cell recordings was compensated to at least 75%. Cells were bathed in a symmetrical potassium solution, and currents were elicited from a holding potential of 0 mV in a 100-ms voltage step to +50 mV. Electrophysiological data were analyzed using pClamp 9.2 (Axon Instruments) and Origin software (5.0; Microcal, Northampton, MA).

Measurement of Mitochondrial Membrane Potential Using Fluorescence Imaging. The mitochondrial membrane potential was measured using rhodamine-123 (Rh-123) fluorescent dye imaging. Rh-123 is a cell-permeant, cationic, fluorescent dye that is readily uptaken by active mitochondria. The uptake of Rh-123 by mitochondria is directly correlated to the membrane potential of mitochondria, suggesting that uptake is probably driven by mitochondrial membrane potential. The linear relationship of the change in Rh-123 fluorescence and mitochondrial membrane potential, the dependence of its uptake by mitochondrial membrane potential, and its low cytotoxicity make this dye an excellent indicator of mitochondrial membrane potential (Chen, 1988). Dissociated neurons were placed in a glass-bottom culture dish and then loaded with 10 µg/ml Rh-123 (Sigma) at room temperature for 10 min. After loading, the neurons were continuously perfused with buffer that contained 140 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 1.0 mM CaCl₂, 10 mM glucose, and 10 mM HEPES. Rh-123 fluorescence images were captured using an inverted microscope with 40x Plan-Neofluar objectives (Axiovert 135; Zeiss) and a silicone intensifier target camera and were recorded on a fluorescence-imaging system (Argus 50/CA; Hamamatsu Photonics, Hamamatsu, Japan). The fluorescence was excited at 490 nm and filtered at 530 nm.

Solutions and Drugs. The composition of the ACSF was 124 mM NaCl, 5 mM KCl, 24 mM NaHCO₃, 1.3 mM MgSO₄, 1.2 mM KH₂PO₄, 2.4 mM CaCl₂, and 10 mM glucose, pH 7.4, bubbled with 95% O₂-5% CO₂. The composition of the standard external solution for DA neuronal recordings was 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, and the pH was adjusted to 7.4 using Tris base. The external solution for HEK-293 cell recordings was composed of 140 mM KCl, 1.2 mM MgCl₂, 2.6 mM CaCl₂, and 5.0 mM HEPES, pH 7.4. The amphotericin B perforated-patch pipette solution contained 130 mM potassium gluconate, 10 mM KCl, 5 mM MgCl₂, and 10 mM HEPES, pH 7.2 (using Tris-OH). The liquid junction potential, calculated using Clampex 9.2 (Axon Instruments), was 14 mV and corrected post hoc. Amphotericin B was dissolved in dimethyl sulfoxide (40 mg/ml), and the stock solution was diluted with internal (patch-pipette) solution to achieve a final concentration of 200 to 300 µg/ml just before use. The pipette solution for conventional whole-cell recordings contained 107 mM KCl, 1.2 mM MgCl₂, 1.0 mM CaCl₂, 10 mM EGTA, 5.0 mM HEPES, and 3.0 mM MgATP, pH 7.3. Pronase was purchased from Calbiochem-Novabiochem Co. (La Jolla, CA); rotenone, tolbutamide, caffeine, thermolysin, amphotericin B, and Lucifer yellow were purchased from Sigma. All other chemicals were purchased from Tocris Cookson, Inc. (Ballwin, MO), with the exception of iptakalim, which was a gift from Dr. H. Wang (Institute of Pharmacology and Toxicology, Beijing, People's Republic of China).

	Results

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Functional pm-K_ATP Channels in Identified DA Neurons. Figure 1, A and B, demonstrate basic electrophysiological properties of dissociated DA neurons: both regular spontaneous action potential firing at 1 to 3 Hz and sensitivity to dopamine (Fig. 1Aa), large after-hyperpolarization potentials and long duration of individual action potential spikes (>2 ms at a 50% level of amplitude, Fig. 1Ab), and hyperpolarization-induced currents (Fig. 1Ba, H-current) or a "sag"-like membrane potential change (Fig. 1Bb) after injection of a hyperpolarizing current. To further confirm the DA phenotype of a neuron following patch-clamp recording, in some experiments the neuron (Fig. 1Ca) was loaded with Lucifer yellow (0.5 mg/ml in the pipette solution) after recording (Fig. 1Cb) by converting to whole-cell configuration from perforated-patch recording and then repetitive hyperpolarizing pulses were applied (5 mV, 0.5 Hz for 2 min). The loaded neuron was then fixed and immunolabeled for TH (Fig. 1Cc). These results indicate that freshly isolated large- or medium-sized neurons from rat SNc displayed electrophysiological, immunohistochemical, and pharmacological properties characteristic of DA neurons.

To examine functional K_ATP channels in single DA neurons, perforated patch-clamp recordings were employed. In current-clamp mode, application of the SUR1-selective KCO diazoxide hyperpolarized the membrane and reduced spontaneous action potential firing (Fig. 1D, n = 6), whereas the SUR2B-containing KCO cromakalim (Avshalumov et al., 2005) exhibited little effect on action potential firing (Fig. 1E), and similar insignificant results were obtained from five other neurons in four experiments. Before and during exposure to KCOs, the frequency of spontaneous action potential firing was 2.0 ± 0.1 and 0.9 ± 0.1 Hz for diazoxide (p < 0.01, n = 6) and 1.5 ± 0.1 and 1.6 ± 0.1 Hz for cromakalim (p > 0.05, n = 6), respectively. Figure 1F shows that rotenone (100 nM) induced gradual membrane hyperpolarization, which was accompanied by a reversible abolishment of spontaneous action potential firing. Similar results were obtained from 10 neurons. To determine whether rotenone-induced hyperpolarization was mediated through the opening of pm-K_ATP channels, the classic K_ATP channel blocker tolbutamide was applied in each case, and the results demonstrate that 100 µM tolbutamide completely reversed rotenone-induced hyperpolarization (Fig. 1F). Because it is known that neuronal K_ATP channels are mostly composed of Kir6.2 and SUR1 and/or SUR2B subunits (Liss et al., 1999a, 2005), these results suggest that functional pm-K_ATP channels (diazoxide- and tolbutamide-sensitive but cromakalim-insensitive) in rat SNc DA neurons are probably composed of Kir6.2 and SUR1 subunits.

Protein Expression of SUR1 and SUR2B Subunits of K_ATP Channels in SNc DA Neurons. To explore the expression of SUR1 and/or SUR2B subunits of K_ATP channels in dissociated rat SNc DA neurons, double immunocytochemical staining using selective SUR1 and SUR2B antibodies was applied. Figure 2A represents an example of double staining in dissociated neurons using SUR1 and SUR2B antibodies ("SUR1" and "SUR2B," in the presence of both subunit primary antibodies) and shows a SUR1-positive (red) but SUR2B-negative reaction. In 24 dissociated neurons from four experiments, 20 neurons showed a SUR1-positive reaction (83%), and four neurons showed a SUR1-negative reaction (17%), whereas no SUR2B-positive reaction was observed. The bottom row of Fig. 2A shows a negative reaction using only secondary antibodies to SUR1 and SUR2B (SUR1- and SUR2B-, in the absence of both subunit primary antibodies). From four additional experiments, double staining was applied using either TH and SUR1 (Fig. 2B, top row) or TH and SUR2B (Fig. 2B, bottom row) antibodies. The results demonstrate that 83 of 90 TH-positive neurons expressed SUR1 (Fig. 2B, green), whereas only seven of 61 TH-positive neurons expressed SUR2B. These results suggest that DA neurons acutely dissociated from rat SNc mainly express the SUR1 subunit together with the Kir6.2 subunit to form functional K_ATP channels.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Immunocytochemical staining to identify SUR1 and SUR2B subunits of KATP channels in SNc DA neurons. A, top row, example of double staining using specific SUR1 and SUR2B antibodies. Phase, phase contrast photo-picture; SUR1, in the presence of SUR1 primary antibody; SUR2B, in the presence of SUR2B primary antibody; SUR1-, in the absence of SUR1 primary antibody; SUR2B-, in the absence of SUR2B primary antibody. B, double staining for TH and SUR1 was applied and showed that TH-positive neurons (red) exhibited a SUR1-positive (green) but a SUR2B-negative reaction.

Iptakalim Failed to Open pm-K_ATP Channels. In identified DA neurons, iptakalim, at a concentration of 3 µM, exhibited little effect on spontaneous action potential firing (Fig. 3A). However, 100 µM iptakalim increased spontaneous action potential firing in three of six DA neurons (data not shown), whereas 300 µM iptakalim increased spontaneous action potential firing in all of the tested DA neurons (Fig. 3B). Before and during exposure to iptakalim, the frequency of spontaneous action potential firing was 2.1 ± 0.2 and 1.9 ± 0.2 Hz for 3 µM (p > 0.05, n = 4), 2.1 ± 0.2 and 2.3 ± 0.1 Hz for 100 µM (p > 0.05, n = 6), and 1.6 ± 0.2 and 2.9 ± 0.2 Hz for 300 µM (p < 0.01, n = 6), respectively. Figure 3C shows that tolbutamide (300 µM), a selective SUR1-containing K_ATP channel blocker, also increased DA neuron firing frequency from 2.2 ± 0.1 to 2.7 ± 0.3 Hz (p < 0.05, n = 4). Figure 3D shows that 300 µM iptakalim increased DA firing frequency. These results suggest that iptakalim failed to directly open pm-K_ATP channels in single DA neurons acutely dissociated from rat SNc. Instead, iptakalim, at relatively high concentrations, closed pm-K_ATP channels.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Effects of iptakalim on DA neuronal activity. In this and the following figures, Ipt is iptakalim. A, low concentration (3 µM) of iptakalim showed little effect on DA neuron action potential firing. B, high concentration (300 µM) of iptakalim increased DA neuron action potential firing. C, the KATP channel blocker tolbutamide (300 µM) similarly increased DA neuron action potential firing compared with iptakalim. Horizontal dashed line in A to C, resting membrane potential. D, frequency histogram shows that 300 µM iptakalim increased DA neuron firing frequency. *, p < 0.05, **, p < 0.01; ***, p < 0.001.

Iptakalim Reversed pm-K_ATP Channel-Mediated Hyperpolarization. In general, K_ATP channels expressed in central neurons are maintained at a closed status under physiological conditions (Busija et al., 2004). However, in midbrain slices, the K_ATP channel blocker glibenclamide increases DA neuronal firing frequency (Avshalumov et al., 2005), suggesting a spontaneous background opening of K_ATP channels occurs using in vitro preparations. Therefore, the above results (i.e., Fig. 3, B and D) suggest that iptakalim-induced increase of neuronal firing may be mediated through an inhibition of such background K_ATP channel activity. To determine whether iptakalim closes pm-K_ATP channels, we examined the effects of iptakalim on rotenone-induced K_ATP channel opening using current-clamp recordings. As shown in Fig. 4A, 100 nM rotenone-induced membrane hyperpolarization was reversed, action potential firing was completely restored by tolbutamide (300 µM), and iptakalim (100 µM) showed similar restorative effects on action potential firing, whereas other KCOs, such as pinacidil (100 µM), diazoxide (100 µM), and P1075 (100 µM), failed to reverse rotenone-induced hyperpolarization. After spontaneous action potential firing recovery after several minutes of washout of rotenone, the KCO diazoxide hyperpolarized DA neurons, which was reversed by iptakalim as well. Similar results were obtained from five other neurons. These results suggest that iptakalim, in particular at high concentrations, probably functions as a pm-K_ATP channel blocker. Figure 4B shows that iptakalim reversed rotenone-induced hyperpolarization in a concentration-dependent manner.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Iptakalim reversed rotenone-induced DA neuron hyperpolarization. A, under current-clamp recording configuration, 100 nM rotenone-induced hyperpolarization was reversed by both tolbutamide (Tol, 300 µM) and iptakalim (Ipt, 100 µM) but not by pinacidil (Pina, 100 µM) or P1075 (100 µM). Using this same neuron, diazoxide (DZX)-induced hyperpolarization was also reversed by iptakalim (100 µM). B, iptakalim reversed rotenone-induced hyperpolarization in a concentration-dependent manner. Horizontal dashed line in A and B, resting membrane potential.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Iptakalim suppressed rotenone-induced outward current. A, under voltage-clamp recording configuration, rotenone induced a slow outward current, which was suppressed by iptakalim in a concentration-dependent manner. Horizontal open bar, exposure to 300 µM tolbutamide. Filled bar, exposure to iptakalim at a concentration of 300 (Aa), 30 (Ab), and 3 (Ac) µM. Ad, effects of iptakalim in the absence of rotenone. Caffeine (10 mM) was applied to induce outward current as a control and then different concentrations of iptakalim were applied at 3-min intervals and did not show any detectable current response. B, bar graph compares the effects of tolbutamide and iptakalim on rotenone-induced currents. Tolbutamide (300 µM)-induced suppression was normalized to 100%. Vertical bars, S.E. **, p < 0.01.

Iptakalim Suppressed Kir6.2/SUR1 K_ATP Channels Heterologously Expressed in Cultured HEK-293 Cells. Data presented thus far suggest that the majority of functional K_ATP channels in rat SNc DA neurons seem to be composed of Kir6.2 and SUR1 subunits and that iptakalim failed to open, but rather likely closed, these K_ATP channels. To further confirm these possibilities, we examined the effects of iptakalim on Kir6.2/SUR1 K_ATP channels heterologously expressed in cultured HEK-293 cells. A depolarizing pulse from 0 to +50 mV (100-ms duration) was applied to a HEK-293 cell transfected with Kir6.2 and SUR1 subunits at a holding potential of 0 mV under a symmetrical internal and external K⁺ (140 mM) condition, and only a tiny leak current was observed (Fig. 6A). Using the same experimental protocol, bath-applied iptakalim (100 µM) showed no significant effects on membrane current (Fig. 6B), but diazoxide (200 µM), on the other hand, induced a large outward current (Fig. 6C), which was blocked by glibenclamide (10 µM), a K_ATP channel blocker (data not shown). These data suggest that diazoxide opened Kir6.2/SUR1 K_ATP channels. Coapplication of 200 µM diazoxide and 300 µM iptakalim (Fig. 6D) or 500 µM iptakalim (Fig. 6E) reduced diazoxide-induced current. In addition, our preliminary results showed that both diazoxide (200 µM, n = 6) and iptakalim (100 µM, n = 6) did not show detectable current induction in wild-type HEK-293 cells (data not shown). Figure 6F summarizes the results from four cells and shows that iptakalim alone did not enhance membrane current; instead, it suppressed diazoxide-induced membrane current. These results support the notion that iptakalim did not open, but rather closed, Kir6.2/SUR1 K_ATP channels in SNc DA neurons.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Effects of iptakalim on Kir6.2/SUR1 KATP channels heterologously expressed in HEK-293 cells. A representative leak current in a transfected cell by a depolarizing pulse (A) was not enhanced by exposing the cell to 100 µM iptakalim for 30 s (B) but was enhanced by 200 µM DZX (C) through the opening of KATP channels. B and C, thin traces, leak current; thick traces, resulting current from drug application. Coexposure to DZX plus 300 µM iptakalim (D) or 500 µM iptakalim (E) suppressed DZX-induced current potentiation. F, bar graph summarizes the effects of iptakalim on Kir6.2/SUR1 KATP channels and DZX-induced current enhancement from four cells. *, p < 0.05; **, p < 0.01.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Iptakalim failed to open mitochondrial KATP channels in SNc DA neurons. The mitochondrial membrane potential was indicated by the intensity of the fluorescent dye Rh-123. All Rh-123 intensity changes were subtracted by background intensity. In the same-monitored DA neuron (A), iptakalim failed to alter Rh-123 intensity even at a concentration of 300 µM (Bb), but both diazoxide (300 µM, Bc) and rotenone (100 nM, Bd) increased Rh-123 intensity, which is a reflection of depolarization of mitochondrial membrane potential. C, bar graph summarizes the effects of iptakalim, diazoxide, and rotenone on DA neuron mitochondrial membrane potential. **, p < 0.01.

Iptakalim Failed to Prevent Rotenone-Induced Mitochondrial Depolarization. Because iptakalim reversed rotenone-induced pm-K_ATP channel-mediated hyperpolarization, we examined whether iptakalim also prevents rotenone-induced mitochondrial depolarization. As shown in Fig. 8, 100 µM iptakalim failed to prevent rotenone-induced depolarization (n = 4), suggesting that iptakalim did not block mito-K_ATP channels in rat SNc DA neurons.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Iptakalim failed to prevent rotenone-induced mitochondrial membrane depolarization in DA neurons. A, mitochondrial membrane potential was indicated by the intensity of the fluorescent dye Rh-123. Metabolic stress induced by rotenone (100 nM) increased Rh-123 intensity (from Aa to Ab), reflecting a depolarization of mitochondrial membrane potential. Iptakalim (100 µM) neither depolarized mitochondrial membrane potential (Ac) nor affected rotenone-induced mitochondrial depolarization (Ad). B, fluorescence images from the recorded neuron illustrated in A. Images B, a to d correspond to A, a to d. The intensity indicator is shown at the right, and the red arrow indicates the direction of mitochondrial membrane depolarization.

	Discussion

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Iptakalim, as a cardiovascular KCO, exhibited clear antihypertensive effects in hypertensive animal models that used in vivo and in vitro preparations (Wang, 2003; Wang et al., 2005a). The possible mechanisms underlying its antihypertensive action include the opening of cardiovascular K_ATP channels (Wang, 2003). In primary cultured hippocampal neurons, iptakalim selectively potentiated voltage-activated K⁺, but not Na⁺ or Ca²⁺, currents (Wang et al., 2004). Furthermore, [³H]iptakalim was reported to bind to SUR receptors of K_ATP channels in rat cerebral cortex, hippocampus, and striatum membrane preparations (Wang et al., 2004). Based on these results, it was postulated that iptakalim served as a KCO that may protect cells against hypoxia/ischemia- and neurotoxin-induced neuronal degeneration (Wang et al., 2005a; Yang et al., 2005a,b,c). However, thus far, there is still no direct experimental evidence supporting the hypothesis that iptakalim is able to directly open pm-K_ATP channels and/or mito-K_ATP channels in SNc DA neurons, the neurons that are key targets in both PD patients and PD animal models.

In the present study, we clearly demonstrate that the mitochondrial complex I inhibitor rotenone and the relatively selective SUR1-containing KCO diazoxide opened pm-K_ATP channels in freshly dissociated rat SNc DA neurons using perforated patch-clamp recordings, which is consistent with previous reports (Liss et al., 1999a,b). In addition, using fluorescence-imaging methods, we also show that both diazoxide and rotenone depolarized mitochondrial membrane, perhaps through the opening of mito-K_ATP channels (Tai et al., 2003). However, under our experimental conditions, we failed to observe any opening of either pm-K_ATP channels or mito-K_ATP channels by iptakalim. The specific subunits expressed in SNc DA neurons may contribute to the observed K_ATP channel insensitivity to iptakalim. It has been reported that in the SNc, DA neurons express functional K_ATP channels consisting of Kir6.2 and SUR1 or SUR2B subunits (Liss et al., 1999a; Avshalumov et al., 2005). Recently, Liss et al. (2005) reported that the majority of K_ATP channels expressed in SNc DA neurons of mice (30-day-old) were mainly composed of Kir6.2 and SUR1 subunits. Pharmacologically, SUR1-containing K_ATP channels exhibit high sensitivity to rotenone (Liss et al., 1999a), whereas SUR2-containing K_ATP channels (probably SUR2B) expressed in DA neurons are sensitive to cromakalim (Avshalumov et al., 2005) and exhibit low sensitivity to rotenone (Liss et al., 1999a,b). In the present study, all DA neurons represented herein were sensitive to rotenone but insensitive to the SUR2B-selective KCO cromakalim, suggesting that the K_ATP channel subunits Kir6.2 and SUR1 may be predominantly expressed in rat SNc DA neurons. This conclusion is further supported by immunostaining experiments, in which dissociated rat SNc DA neurons mainly exhibited a SUR1-rather than a SUR2B-positive reaction. It is well known that the subunit combination of Kir6.2/SUR1, which forms functional K_ATP channels, is mainly expressed in pancreatic -cells (Ashcroft and Gribble, 2000; Mannhold, 2004), whereas cardiovascular smooth muscle cells mainly express functional K_ATP channels composed of Kir6.2/6.1 and SUR2A/B (Ashcroft and Gribble, 2000; Mannhold, 2004). This difference in expression of SUR subunits determines both the affinity and efficacy of pinacidil-like (e.g., pinacidil and P1075) KCOs (Ashcroft and Gribble, 1998, 2000; Mannhold, 2004). Based on previous studies, iptakalim seems to open K_ATP channels by binding to the SUR2A and/or SUR2B subunit because iptakalim competed against [³H]P1075 in binding to isolated smooth cells (Wang, 2003), and [³H]iptakalim binding in the central nervous system was displaced by pinacidil or P1075 (Wang et al., 2004). Recently, Wang's laboratory found that in heterologously expressed K_ATP channels in cultured HEK-293 cells, iptakalim opened SUR2A- and SUR2B-, but not SUR1-containing K_ATP channels (Y. P. Chen and H. Wang, unpublished data; Fig. 6B). This implies that iptakalim is incapable of opening SUR1-containing K_ATP channels. Therefore, it is possible that rat SNc DA neurons mainly express Kir6.2 and SUR1 subunits, which may explain why iptakalim was not able to open these K_ATP channels. This interpretation is consistent with previous reports that pinacidil failed to open K_ATP channels composed of Kir6.2 and SUR1 subunits in Xenopus oocytes (Ashfield et al., 1999) and that A10 DA neurons were sensitive to diazoxide but not to pinacidil or lemakalim (Scuvee-Moreau et al., 1997). Moreover, we recently found that iptakalim also failed to open K_ATP channels in rat pancreatic -cells (N. Misaki, S. Suga, X. Mao, Y. F. Lin, M. Wakul, and J. Wu, unpublished data), signifying that iptakalim is incapable of opening SUR1-containing K_ATP channels. Therefore, it is likely that iptakalim opens SUR2A- and/or SUR2B- but not SUR1-containing K_ATP channels.

Instead of opening neuronal K_ATP channels, we surprisingly found that iptakalim closed pm-K_ATP channels expressed in rat SNc DA neurons. The evidence of similar inhibitory effects of iptakalim on transfected Kir6.2/SUR1 K_ATP channels further supports our conclusions. The mechanisms responsible for the iptakalim-induced block of pm-K_ATP channels are unclear, although several explanations are possible. Iptakalim may bind to glibenclamide sites of the SUR1 subunit, thereby altering SUR subunit conformation, which in turn would diminish K_ATP channel opening. Emerging evidence has demonstrated that iptakalim-induced pharmacological effects in the cardiovascular and central nervous systems can be prevented by pretreatment using glibenclamide (Wang et al., 2004, 2005a,b,c; Yang et al., 2005a). Iptakalim and glibenclamide may compete for similar ligand binding sites on the SUR1 subunit. In addition, iptakalim may increase K_ATP channel sensitivity to ATP. It has been reported that some K_ATP channel modulators regulate K_ATP channel activity by altering K_ATP channel sensitivity to ATP (Suga et al., 2001). Finally, iptakalim may directly block K_ATP channels by acting on the Kir6.2 subunit. It is known that some K_ATP channel modulators, such as nicorandil, pinacidil, or glibenclamide, regulate K_ATP channel activity by targeting the regulating subunit SUR (Gribble and Ashcroft, 2000a,b), whereas others (e.g., phentolamine and cibenzoline) directly inhibit the pore-forming subunit Kir6.2 (Proks and Ashcroft, 1997; Mukai et al., 1998). Ultimately, although the underlying mechanisms are unknown, the new discovery that iptakalim blocks pm-K_ATP channels will promote further pharmacological investigation.

The pharmacological significance of iptakalim's block of pm-K_ATP channels is unclear; it seems contradictory to the expectation that iptakalim should open pm-K_ATP channels and, in turn, protect DA neurons against chemical stress-induced neurodegeneration. However, new experimental evidence and concepts have recently emerged that K_ATP channels promote DA neuronal degeneration. For example, in Kir6.2 knockout mice, rotenone-induced DA neuronal death was significantly prevented, suggesting that inhibition of K_ATP channels may play an important role in neuroprotection (Liss et al., 2005). From this point of view, the discovery of the blocking effects on Kir6.2/SUR1 K_ATP channels by iptakalim has led to a new direction in the study of the pharmacological mechanisms of its neuroprotective effects in various PD animal models using in vivo and in vitro preparations.

The present results do not support the hypothesis that iptakalim protects SNc DA neurons against chemical stress-induced neurotoxicity by directly opening pm-K_ATP channels and/or mito-K_ATP channels in the somatodendrite of DA neurons, which raises caution against the simple interpretation that effective neuroprotection induced by iptakalim against metabolic stress using in vivo animal models is via the opening of pm-K_ATP channels and/or mito-K_ATP channels in SNc DA neurons. However, our data cannot exclude other possible protective effects of iptakalim associated with the opening of K_ATP channels expressed in elements other than DA neurons themselves.

	Acknowledgements

 
We thank Kevin Ellsworth for assistance in preparing the manuscript and Hai Wang (Beijing Institute of Pharmacology and Toxicology, People's Republic of China) for kind support by providing iptakalim as a gift.

	Footnotes

 
This work was supported by the Women's Board Foundation of the Barrow Neurological Institute.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.106286.

ABBREVIATIONS: PD, Parkinson's disease; pm, plasma membrane; mito, mitochondrial; DA, dopaminergic; SNc, substantia nigra pars compacta; SUR, sulfonylurea; HEK, human embryonic kidney; TH, tyrosine hydroxylase; PBS, phosphate-buffered saline; Rh-123, rhodamine-123; P1075, N-cyano-N''-(1,1,-dimethylpropyl)-N''-3-pyridylguanidine; DZX, diazoxide; ACSF, artificial cerebrospinal fluid.

Address correspondence to: Dr. Jie Wu, Neurophysiology Laboratory, Neurology Research, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013-4496. E-mail: Jie.Wu{at}chw.edu

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