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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
Submitted 30 September 2004 ; accepted in final form 9 August 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-subunit of the BK channel (BK-), both - and 
1-subunits (BK-1), or both - and 4-subunits (BK-4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-1 within a similar concentration range. Because methAEA could potentiate BK-, BK-1, and BK-4 with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener
-subunit and accessory -subunits (1–4) arranged in tetramers (24). Each -subunit interacts with the NH2-terminal region of the -subunit and regulates the activity of the -subunit by changing Ca2+ and voltage sensitivity and/or channel kinetics (10, 11). Although only one major type of -subunit with splice variants has been found, several subtypes of -subunits whose 1- and 4-isoforms are abundantly expressed in smooth muscle and central nervous system (CNS), respectively, have been cloned and may be responsible for the tissue-specific characteristics of BK channels (4, 36, 44). Synthetic compounds such as NS-1619 and BMS-204352 are activators of the BK- subunit, whereas dehydrosoyasaponin I (14), 17-estradiol (37, 3), and tamoxifen (9) act on the BK 1-subunit. Agents that enhance BK channel activity (BK channel openers) may be effective in protecting neurons from damage after an ischemic stroke and/or in suppressing excess activity of smooth muscle tissues (23, 35). 17-Estradiol (37), arachidonic acid (AA) (21), epoxyeicosatrienoic acids (EETs) (13), and dihydroxyeicosatrienoic acids (25) may be endogenous BK channel openers, and some transmitters and hormones also can enhance BK channel activity via activation of protein kinases.
Anandamide (AEA), an endogenous cannabinoid, is an effective vasorelaxant in many types of blood vessels (32) and therefore can significantly reduce blood pressure as do other cannabinoids (38, 39). These responses are mediated mainly by the cannabinoid receptor CB1 as shown previously using selective CB1 antagonists (29, 38). However, a substantial part of the relaxation caused by cannabinoids is resistant to treatment with both CB1 and CB2 antagonists, suggesting that certain receptor-independent mechanisms are involved in the relaxation. Although a major breakthrough showed that AEA activates vanilloid receptors (VR1) in perivascular sensory nerves, a potent AEA-induced relaxation remained unchanged in the presence of CB1/CB2 and VR1 antagonists (49). In rat coronary and mesenteric arteries, this CB- and VR1-independent relaxation was sensitive to BK channel blockers, indicating that cannabinoids act on BK channels as "a potential endogenous BK channel opener" (30, 43). This fascinating hypothesis has been examined in a few studies (33, 49; for review, see Ref. 32) but, because of the multifunctional activity of AEA, has not been proved conclusively yet. For example, AEA directly blocks two-pore-type K+ channels (twin weak inwardly rectifying K+-related, acid-sensitive K
channels; Ref. 26) and T-type voltage-dependent Ca2+ channel currents (5), but it activates VR1.
The present study was undertaken to determine whether cannabinoids can activate BK channel currents expressed in human embryonic kidney (HEK)-293 cells in which the -subunit of the BK channel (BK-), both - and 1-subunits (BK-1), or both - and 4-subunits (BK-4) were heterologously expressed. We found that both AEA and a stable analog, methanandamide (methAEA), have a potent BK channel-opening action.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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(double-digested with KpnI/XbaI) and BK-1 and BK-4 (double-digested with EcoRI/XbaI) were ligated into the mammalian expression vectors pcDNA3.1(+) and pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad, CA), respectively, using the TaKaRa ligation kit, version 1 (TaKaRa) (45). HEK-293 cells were obtained from the Health Science Research Resources Bank (Tokyo, Japan) and maintained in MEM (GIBCO-BRL, Rockville, MD) supplemented with 10% heat-inactivated FCS (JRS Biosciences, Lenexa, KS), penicillin (100 U/ml; Wako, Osaka, Japan), and streptomycin (100 µg/ml; Meiji Seika, Tokyo, Japan). Stable expression of BK- and BK- was achieved using calcium phosphate coprecipitation transfection techniques. Cells resistant to G418 (1 mg/ml; GIBCO-BRL) and G418/zeocin (0.25 mg/ml; Invitrogen) were selected as those expressing BK- and coexpressing BK-, respectively. Expression of BK- and BK- transcripts was confirmed using RT-PCR. Transfected cell lines were maintained in MEM supplemented with 10% FCS and G418 (0.5 mg/ml). The expression levels of BK- (
90%), BK-1 (80%), and BK-4 (80%) were confirmed using whole cell or inside-out patch-clamp recordings of BK channel currents.
Cell dispersion.
All experiments were carried out in accordance with the guiding principles for the care and use of laboratory animals (the Science and International Affairs Bureau of the Japanese Ministry of Education, Science, Sports and Culture) and also with the approval of the Ethics Committee in Nagoya City University. Male mice (BALB/c) weighing 20–30 g were anesthetized with ether and killed by exsanguination. After opening the chest, we excised an 1.5-cm-long segment of the thoracic aorta. We removed connective tissues and rubbed the inner wall of the vessel with a cotton pad to remove endothelium and then incubated strips of aorta 0.7 cm long in nominally Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) for 5 min; the strips were then placed into solution containing 2 mg/ml collagenase (Amano, Nagoya, Japan) and 1 mg/ml papain (Sigma) for 45 min. After this step, the enzyme-treated strips were mechanically agitated in fresh Ca2+- and Mg2+-free HBSS that did not contain digestion enzymes. Dissociated cells were used within 6 h after cell dispersion. Ca2+- and Mg2+-free HBSS for cell dispersion contained (in mM) 137 NaCl, 5.4 KCl, 0.168 Na2HPO4, 0.44 KH2PO4, 5.55 glucose, and 4.17 NaHCO3 (pH 7.45).
Solutions. The HEPES-buffered solution for electrophysiological recording had an ionic composition of (in mM) 137 NaCl, 5.9 KCl, 2.2 CaCl2, 1.2 MgCl2, 14 glucose, and 10 HEPES. The pH of the solution was adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 140 KCl, 1 MgCl2, 10 HEPES, 2 Na2ATP, and 5 EGTA. The pCa and pH of the pipette solution were adjusted to 6.5 and 7.2, respectively, by adding CaCl2 and KOH, respectively. During recordings of a single BK channel current in the inside-out patch-clamp configuration, the pipette solution contained the HEPES-buffered solution and the bathing solution contained (in mM) 140 KCl, 1.2 MgCl2, 14 glucose, 10 HEPES, and 5 EGTA. Selected pCa levels of the bathing solution were obtained by adding an adequate amount of CaCl2, and pH was adjusted to 7.2 with NaOH. When ATP was included in the bathing solution in the excised inside-out patch configuration, free Mg2+ and Ca2+ concentrations were kept constant.
Electrophysiological experiments.
The patch-clamp techniques were applied to HEK-293 cells and aortic cells using a CEZ-2400 amplifier (Nihon Kohden, Tokyo, Japan). The procedures for electrophysiological recordings and data acquisition and analysis for whole cell recording were described previously (18). Whole cell currents were recorded from each cell, and leakage currents at potentials positive to –60 mV were subtracted digitally, assuming a linear relationship between current and voltage in the range of –80 to –60 mV. Single-channel currents were recorded in the cell-attached or inside-out patch configuration and were analyzed using the software PAT, version 7.0C, developed at the University of Strathclyde (Dr. J. Dempster). The relative open-state probability of channels (NPo) was calculated using the following equation
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for whole cell and 10–15 M for cell-attached patch-clamp and inside-out patch-clamp configurations when filled with the pipette solutions. The series resistance was partly compensated electrically in a whole cell voltage-clamp configuration. Whole cell and single-channel recordings were obtained at room temperature (24 ± 1°C). Ca2+ fluorescence measurements using fura-2. The measurement of changes in intracellular Ca2+ concentration ([Ca2+]i) using fura-2 (Molecular Probes, Eugene, OR) was performed as described previously (45). Before the fluorescence measurements were performed, cells were incubated with 10 µM fura-2 AM in HEPES-buffered solution for 30 min at room temperature. The fluorescence emission was collected from cell clusters using a dichroic mirror (505 nm) and a BA filter (>520 nm). Data collection and analyses were performed using a Ca2+ imaging system (ARGUS-HiSCA; Hamamatsu Photonics, Hamamatsu, Japan). The sampling interval of fura-2 fluorescence measurements was 10 s.
Chemicals. Drugs were obtained from the following sources. AEA, methAEA, and penitrem A were purchased from Sigma-Aldrich (St. Louis, MO); methAEA and cholera toxin (CTX) were obtained from Calbiochem; pertussis toxin (PTX) and PD-98059 were purchased from Funakoshi (Tokyo, Japan); W-7 was obtained from Biomol (Plymouth Meeting, PA); AM251 and JWH133 were purchased from Tocris Cookson (Ellisville, MO); and KT5823 was obtained from Wako (Tokyo, Japan). PD-98059, AM251, JWH133, and KT5823 were dissolved in 100% DMSO and AEA and methAEA were dissolved in 100% ethanol to make the stock solution. Other agents were dissolved in distilled water. The final concentrations of DMSO and ethanol were 0.1% or lower.
Statistics. Data are expressed as means ± SE. The statistical significance between two groups and among multiple groups was evaluated using Student's t-test and multiple comparisons after ANOVA, respectively. Single and double asterisks in the figures indicate P < 0.05 and P < 0.01, respectively.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 under whole cell voltage-clamp conditions. The [Ca2+] in the pipette solution was fixed at pCa 6.5 using a Ca2+-EGTA buffer. Depolarization from –60 to +20 mV induced outward currents in both native HEK-293 cells and HEK-BK-1, but the current density was much higher in the latter (peak current density was 10.92 ± 0.57 pA/pF for HEK-293 cells and 20.73 ± 2.0 for HEK-BK-1, n = 5 and 6, respectively; P < 0.01). Application of 3 µM AEA significantly increased the outward currents in HEK-BK-1 (0.71 ± 0.13 and 2.59 ± 0.36 nA in the absence and presence of AEA, respectively; P < 0.01) (Fig. 1, A and B). This enhancement of outward currents by AEA was inhibited completely by the addition of 1 µM penitrem A, a relatively specific blocker of the BK channel (Fig. 1B) (16), indicating that AEA effectively potentiated BK channel currents in HEK-BK-1 (see also Fig. 3).
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1 (Fig. 2Aa), and the effect on the current-voltage relationship of peak outward currents is summarized in Fig. 2Ab. As shown in Fig. 2B, the outward currents elicited by depolarization from –60 mV in 10-mV increments were enhanced at potentials positive to –30 mV. However, methAEA enhanced BK channel currents in a voltage-independent manner because the increase produced by 3 µM methAEA at each potential was not significantly different (n = 9). In Fig. 3, the concentration-response relationships of cannabinoids are summarized. The peak amplitude of the outward currents at +20 mV in the presence of each concentration of cannabinoids was measured relative to the value obtained just before the application, and pooled data were plotted against concentration of cannabinoids. The outward currents were potentiated by the cannabinoids as follows: from 0.72 ± 0.11 nA to 1.27 ± 0.36 nA (0.3 µM AEA), 2.10 ± 0.58 nA (1 µM AEA), and 2.51 ± 0.35 nA (3 µM AEA) and from 0.81 ± 0.09 nA to 1.35 ± 0.25 (0.3 µM methAEA), 3.00 ± 0.70 (1 µM methAEA), and 3.22 ± 0.84 nA (3 µM methAEA)
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with those on BK-1 and BK-4.
An increase in outward currents by methAEA in HEK-BK-1 or HEK-BK-4 was compared with that in HEK-BK- under the same experimental conditions described in the legends to Figs. 1 and 2. It has been established that coexpression of the 1- and 4-subunits with BK- slowed the activation time course of BK channel currents after depolarization (7, 40). In the present study, the half-activation time of BK channel currents in HEK-BK- after depolarization to +40 mV was significantly shorter than that of HEK-BK-1 and HEK-BK-4 (1.6 ± 0.4 ms in HEK-BK- vs. 21.3 ± 4.5 ms and 23.8 ± 2.6 ms in HEK-BK-1 and HEK-BK-4, respectively, n = 4–6; P < 0.01), indicating that the BK-1 and BK-4 subunits were functionally coexpressed with the -subunit. As shown in Fig. 4, BK channel currents in HEK-BK-, HEK-BK-1, and HEK-BK-4 elicited by depolarization to +20 mV from a holding potential of –60 mV were increased in a concentration-dependent manner by application of methAEA. In Fig. 4D, concentration-response relationships of methAEA-induced potentiation of BK channel currents in HEK-BK-, HEK-BK-1, and HEK-BK-4 are summarized. The peak amplitude of BK channel currents at +20 mV in the presence of each concentration of methAEA was measured relative to the value obtained just before the application, and pooled data were plotted against the concentrations of methAEA. As a control experiment, methAEA was cumulatively applied to native HEK. The outward currents in the presence of 0.3, 1.0, and 3 µM methAEA were significantly increased in HEK-BK- (n = 3–6; P < 0.05), HEK-BK-1 (n = 6–12; P < 0.01), and HEK-BK-4 (n = 4–6; P < 0.01) compared with those in native HEK-293 cells. In contrast, the coexpression of either the 1- or the 4-subunit did not significantly affect the enhancement of BK- channel currents by methAEA (P > 0.05), indicating that the BK -subunits have a minor role in the activation of BK channel currents by cannabinoids.
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channels and the complex with the 1-subunit (BK-1) were examined in the cell-attached and excised inside-out patch configurations to clarify whether direct interaction of cannabinoids with BK channels is involved. The pCa and concentration of K+ in the bathing solution were 5.0 and 140 mM, respectively. The pipette contained normal HEPES-buffered solution (5.9 mM K+). The unitary current amplitude and NPo at a holding potential of +30 mV were 11.17 ± 0.36 pA (n = 5) and 0.0025 ± 0.0011 (n = 10), respectively. Application of 0.3 and 3 µM methAEA to the bathing solution increased the channel activity of BK- (4.14 ± 1.07- and 9.63 ± 1.47-fold, respectively) and of BK-1 (8.04 ± 3.6- and 22.1 ± 9.8-fold, respectively) (Fig. 5A). On the other hand, the single-channel conductance of BK- measured in the cell-attached patch configuration was 155.4 ± 32.0 and 156.5 ± 19.8 pS (n = 2–11; P > 0.05) in the absence and presence of 0.3 µM methAEA, respectively; that of BK-1 was 151.6 ± 8.7 pS and 152.6 ± 4.6 pS (n = 2–6; P > 0.05), indicating that methAEA does not affect BK channel conductance.
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and BK-1 channel currents were therefore examined in an excised inside-out patch configuration (Fig. 5B). The pCa in the bathing solution and the holding potential were 6.5 and +30 mV, respectively. The K+ concentrations in the bathing and pipette solutions were 140 and 5.9 mM, respectively. Application of 0.3 and 3 µM methAEA did not affect the Po of BK- but rather tended to reduce it (0.72 ± 0.11- and 0.78 ± 0.2-fold of control in the presence of 0.3 and 3 µM methAEA, respectively, n = 9; P > 0.05) (Fig. 5Ba). Consistently, BK-1 channel currents recorded under the same experimental conditions (cell-free, excised inside-out patch-clamp configuration) were not affected by 0.3 and 3 µM methAEA (Fig. 5Bc). Even in the presence of ATP on the cytoplasmic side of the membrane, the activity of BK-1 was not potentiated by 3 µM methAEA (Fig. 5Bc). Moreover, the elevated BK channel activity produced by methAEA in the cell-attached patch-clamp configuration disappeared immediately after the establishment of the excised inside-out patch (data not shown; n = 3). Taken together, these results suggest that methAEA activates BK channel currents without direct interaction with the channel but that certain factors in the cytosol may be essential for activation.
Mechanisms involved in the activation of BK channel currents by cannabinoids.
AEA can activate cannabinoid receptors CB1 and CB2 as an endogenous agonist, and methAEA is a relatively selective agonist for the CB1 receptor (1). In vascular endothelial cells, the activation of CB1 elevates [Ca2+]i (27). Thus we examined the effects of methAEA on [Ca2+]i in HEK-BK- and HEK-BK-1 (Fig. 6). Cumulative application of methAEA in a concentration range between 0.3 and 10 µM did not affect [Ca2+]i in native HEK-293 cells, HEK-BK-, or HEK-BK-1, while 10 µM ACh induced an increase in [Ca2+]i in each cell type. The larger increase in [Ca2+]i by 10 µM ACh in HEK-BK- and HEK-BK-1 (Fig. 6B) may indicate that ACh-induced hyperpolarization resulting from BK channel activation potentiates Ca2+ entry through cation channels endogenously expressed in HEK-293 cells (45).
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1 to 5 µM AM251 for 30 min did not affect methAEA-induced potentiation of BK channel currents (0.57 ± 0.15 nA and 1.43 ± 0.32 nA in the absence and presence of methAEA, respectively) (Fig. 7A). In addition, BK channel currents in HEK-BK-1 were not potentiated by the application of 1 µM JWH133, a selective and potent agonist of the CB2 receptor (0.39 ± 0.07 nA and 0.38 ± 0.09 nA in the absence and presence of JWH133, respectively, n = 4; P > 0.05). These currents were effectively enhanced, however, by the addition of 3 µM methAEA (3.4 ± 0.3 nA, n = 4), thus excluding the possibility of the involvement of CB1 and CB2 receptors in methAEA-induced potentiation of BK channel currents. The pretreatment of HEK-BK-1 with 400 ng/ml CTX or 200 ng/ml PTX for 24 h, which affect Gs or Gi proteins, respectively, had no effect on methAEA-induced potentiation of BK channel currents (PTX, 0.63 ± 0.22 nA and 1.49 ± 0.66 nA in the absence and presence of methAEA, respectively; CTX, 1.03 ± 0.22 nA and 3.00 ± 0.83 nA in the absence and presence of methAEA, respectively) (Fig. 7A). In the presence of 100–300 µM GTP in the bathing solution, the activity of the BK channel in the inside-out patch-clamp configuration was not affected by 3 µM methAEA added to the pipette solution (0.98 ± 0.21 control, n = 5; P > 0.05). These results strongly suggest that G proteins, including Gs and Gi, which have been shown to modulate BK channel activity in previous studies, are not direct targets of methAEA.
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Effects of methAEA on macroscopic BK channel currents in mouse aortic myocytes. For physiological relevance, it is important to demonstrate cannabinoid-induced potentiation in a cell type with native BK channels. The effects of methAEA on BK channel currents were thus examined in myocytes dispersed from mouse aorta (Fig. 8). The [Ca2+] in the pipette solution was fixed at pCa 6.5. To block voltage-dependent Ca2+ channel currents, 50 µM Cd2+ was present in the bathing solution. Depolarization from –60 to +20 mV induced outward currents, and application of 1 µM methAEA significantly increased the currents (228.8 ± 41.6 pA and 510.0 ± 77.5 pA in the absence an presence of methAEA, respectively; P < 0.05) (Fig. 8). This enhancement of outward currents by methAEA was effectively inhibited by addition of 1 µM penitrem A (39.3 ± 3.5 pA; P < 0.01), suggesting that cannabinoids are potential endogenous BK channel openers.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 or BK-4 with the -subunit did not significantly affect the cannabinoid-induced potentiation of current through the BK- subunit. The channel activity of BK- or the complex with BK- in the excised patch was not susceptible to cannabinoids. The potentiation of BK channels by cannabinoids is not mediated by the stimulation of CB1 and CB2 receptors, the activation of Gs and Gi proteins, or the direct activation of the BK -subunit, but by unknown cytosolic factors excluding CaM, MAPK, and G kinase. Cannabinoids potentiated BK channels. Cannabinoid-induced potentiation of BK channel activity is electrophysiologically characterized as follows: 1) potentiation occurs in the cell-attached patch-clamp mode but not in the excised inside-out patch-clamp configuration, 2) slow onset is followed by a gradual increase, 3) there is no change in BK channel conductance, and 4) there is voltage-independent enhancement.
In contrast to the direct activation of BK channels by fatty acids, including AA, in excised patches (21, 6), we have clearly shown that cannabinoids had stimulatory effects on BK channel activity only in whole cell and on cell patch-clamp configurations. Similar dissociations of effectiveness among patch-clamp configurations have been reported in BK channel activation by EETs (13). The presence of GTP in the bathing solution, however, recovered EET-induced activation of BK channels. These findings suggested that a G protein has an obligatory role in the activation of BK channels by EET and are consistent with the disappearance of potentiation of EET-induced BK channels by pretreatment with CTX (13). In the present study, despite the presence of GTP in the bathing solution, methAEA in the pipette solution failed to activate the BK channel in the inside-out patch-clamp configuration. In whole cell recordings, the onset of BK channel current potentiation by EET started 3–5 min after the application. In contrast, an immediate potentiation was observed within 1 min after the application of AA and NS1619, which also are effective in excised patches (6, 21, 25). Consistently, protein Gs and the subsequent activation of PKG mediate the potentiation of the BK channel by EET (13). The onset of BK channel current potentiation by AEA (Fig. 1) and methAEA was also gradual. These analyses provide a line of evidence indicating that certain soluble factors in the cytosol are essential for activation.
Endogenous and synthesized BK channel openers are known to enhance channel activity by increasing the probability, but not by increasing single-channel conductance, and methAEA follows this pattern as well. Regarding the voltage dependence of BK channel openers, BMS-204352 (15), 17-estradiol (37), and tamoxifen (9) show potentiating effects only at positive potentials. In contrast, methAEA as well as NS-1619, nordihydroguaiaretic acid (46), and pimaric acid (19) enhance BK channel current at potentials positive to –30 mV in a voltage-independent fashion. This feature of methAEA is important with respect to the regulation of resting membrane potential by BK channel activation. Although the sensitivity of BK channels to Ca2+ is considered to be affected by these BK channel openers (10, 15, 19, 25), effects of cannabinoids on Ca2+ sensitivity could not be examined systematically in the present study, because cannabinoids were ineffective on BK channel activity recorded in the cell-free excised patch-clamp configuration, in which [Ca2+]i can be controlled precisely. BK channel currents were effectively potentiated by methAEA when [Ca2+]i was kept at 100 nM Ca2+ in the whole cell recording mode.
BK - and -subunits in methAEA-induced potentiation of BK channel currents.
In the present study, we have shown that BK -subunits were not involved in methAEA-induced potentiation of BK channel currents. In addition, the following evidence supports the hypothesis that methAEA does not interact directly with either the BK - or -subunit. The efficacy of activation of BK channels was comparable among HEK-BK-, HEK-BK-1, and HEK-BK-4; external application of methAEA activated both BK- and BK-1, even in the cell-attached patch-clamp configuration, in which direct access of methAEA to the channel was impossible; and methAEA failed to activate both BK- and BK-1 in the excised inside-out patch-clamp configuration. A primary target of nordihydroguaiaretic acid and pimaric acid into BK channels is the BK -subunit itself; the efficacy of these compounds in activating BK channel currents composed of the BK -subunit alone is comparable to the activation of currents coexpressing BK - and BK 1-subunits. In addition, these BK channel openers effectively activated BK- channels in the excised inside-out patch configuration (19, 46), and it is likely that BMS-204352 interacts mainly with the BK -subunit (15). In contrast, dehydrosoyasaponin I, 17-estradiol, and tamoxifen increase BK channel currents only when the BK -subunit is coexpressed with BK-1, suggesting that these compounds bind to the BK-1 subunit (9, 14, 37). The BK 1- and 4-subunits are expressed predominantly in smooth muscle tissues and the CNS, respectively (22, 10, 3). The present results suggest the possibility that cannabinoids activate BK channel currents in organs in which BK channels are expressed, although additional experiments with special attention to the identification of cytosolic factors mediating the BK channel activation are required.
Mechanisms underlying the activation of BK channels by cannabinoids.
It is unlikely that AEA and methAEA potentiate BK channel currents via the activation of cannabinoid receptors expressed in HEK-293 cells, because 1) treatment of cells with a potent blocker of CB1 receptor, AM251 (28), had no effect on methAEA-induced potentiation of BK channel currents; 2) application of JWH133, a potent and selective agonist of CB2 receptor, failed to activate BK channel currents; and 3) modulation of G proteins by PTX and CTX did not affect potentiation. BK channel currents in human umbilical vein endothelial cells are potentiated via the activation of a non-CB1/non-CB2 receptor that couples to stimulation of G kinase through the PTX-sensitive G protein (2). Although it has been reported that AEA elevates [Ca2+]i in vascular endothelial cells (27, 47), methAEA had no effect on Ca2+ fluorescence signals in native HEK-293 cells, HEK-BK-, or HEK-BK-1 in the present study (Fig. 6). However, it cannot be ruled out that non-CB1/non-CB2 receptor stimulation and subsequent signal transduction, in which the activation of G proteins insensitive to both CTX and PTX is involved, are included in the response to cannabinoids in HEK-293 cells.
In many types of cells, cannabinoids modulate cellular functions in a receptor-independent manner, mostly because of metabolic production of bioactive derivatives and/or direct interaction with signaling molecules (8, 41). It has been shown that EETs, metabolites of AEA, can activate BK channel currents via direct activation of CTX-sensitive Gs protein (13). It is unlikely, however, that EET has a central role in the potentiation of BK channel currents by cannabinoids, because methAEA, a stable analog of AEA, had potency comparable to AEA (Fig. 3) and even higher potency after the currents were enhanced by direct activation of the Gs protein by CTX [current amplitudes of BK-1 at +30 mV with and without CTX were 1.03 ± 0.22 nA (n = 5) and 0.54 ± 0.08 nA (n = 8), respectively; P < 0.05]. The Gs protein is therefore a positive regulator of the BK channel but may play only a minor role in cannabinoid-induced potentiation.
Cytosolic factors that may mediate the cannabinoid-induced potentiation of the BK channel remain to be determined. Because the application of W-7, KT5823, or PD-98059 did not change the potentiation, CaM, MAPK, and G kinase may not be involved in the underlying mechanisms. Moreover, application of ATP to the cytoplasmic side of the BK channels did not recover the response to methAEA in the excised inside-out patch-clamp configuration, suggesting that ATP-dependent phosphorylation as well as ATP itself may have minor roles in the cannabinoid-induced potentiation of the BK channel.
Physiological roles of BK channel activation by cannabinoids. The present study provides the first direct evidence that cannabinoids activate BK channel currents in vascular smooth muscle cells as well as in HEK-293 cells, in which the channels are heterologously expressed. It has been proposed that AEA hyperpolarizes vascular smooth muscles as an endothelium-derived hyperpolarizing factor (31, 32). In fact, AEA causes hyperpolarization and relaxation in some vascular smooth muscles that are sensitive to BK channel blockers (31, 42). Results that contrast with these observations also have been reported (12, 48). Although the reason for the discrepancy is not clear, the present results strongly support the hypothesis that cannabinoids are potential hyperpolarizing factors mediated by activation of BK channels expressed in vascular smooth muscles and may contribute to the regulation of vascular tone.
Because cannabinoids activate BK channel currents regardless of accompanying BK -subunit subtypes, it may be possible that some of the effects induced by endogenous cannabinoids in the CNS are mediated by the activation of BK channels in neurons, where 4- and 3-isoforms are predominantly expressed. A splice variant of BK-3, which produces rapid inactivation properties in BK channels, has been correlated with idiopathic epilepsy (17). Decreased BK channel current by splice variant-induced inactivation may be responsible for hyperexcitability in the CNS. It also has been reported that leptin, an opener of BK channels, suppresses epileptiform-like electrical activity in rat hippocampus neurons (34). Opening of BK channels by cannabinoids may reduce membrane excitability in the CNS as well as in vascular organs and therefore may be involved in physiological regulation of CNS and circulation function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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-ratio in fluorescence intensity of fura-2 after application of methAEA and ACh. Each agent was also applied to native HEK-293 cells as a control. Open, closed, and hatched columns indicate the 
