Articles in PresS. Am J Physiol Cell Physiol (August 17, 2005). doi:10.1152/ajpcell.00482.2004
C-00482-2004 by Sade et al. R-2 American Journal of Physiology Cell Physiology C-00482-2004 Revised version 2
Activation of large-conductance Ca2+ activated K+ channels by cannabinoids
Hiroko Sade, Katsuhiko Muraki, Susumu Ohya, Noriyuki Hatano and Yuji Imaizumi
Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University
3-1 Tanabedori, Mizuhoku, Nagoya 467-8603 Japan
Running head: BK channel and cannabinoids
Correspondence to Yuji Imaiuzmi, Ph.D. Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603 Japan Tel/Fax: 81(country code)-52(area code)-8363431 Email:
[email protected]
1 Copyright © 2005 by the American Physiological Society.
C-00482-2004 by Sade et al. R-2 Abstract We examined the effects of the cannabinoid anandamide (AEA) and its stable analogue, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using HEK293 cells, in which the α-subunit of the BK channel (BKα), both α and β1 (BKαβ1), or bothα and β4(BKαβ4) subunits were heterologously expressed. In a whole-cell, voltageclamp configuration, each cannabinoid activated BKαβ1 in a similar concentration range. Because methAEA could potentiate BKα, BKαβ1, and BKαβ4 with similar efficacy, the β subunits may not be involved in 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 HEKBKα and HEKBKαβ1 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 application of a elective CB2 agonist (JWH133). Inhibitors of calmodulin, protein kinase G, and MAP kinases (W7, KT5823, and PD98059) 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 factor(s) 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, where BK channels are highly expressed.
Keywords: large-conductance Ca2+-activated K+ channel, anandamide, BK channel opener
2
C-00482-2004 by Sade et al. R-2 Introduction Ca2+-activated K+ (BK) channels consist of channel-forming α subunits and accessory β subunits (β1–β4) arranged in tetramers (24). Each β subunit interacts with the N-terminal region of the α subunit and regulates the activity of the α subunit by changing Ca2+ and voltage sensitivity and/or channel kinetics (10, 11). Although only one major type of α subunit with splice variants has been found, several subtypes of β subunits whose β1 and β4 isoforms are abundantly expressed in smooth muscle and central nervous system, 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 BKα subunit, while dehydrosoyasaponin-I (14), 17β-estradiol (37, 3), and tamoxifen (9) act on the BKβ1 subunit. Agents that enhance BK channel activity (BK channel openers) may be effective in protecting neurons from damage following an ischemic stroke and/or suppressing excess activity of smooth muscle tissues (23, 35). 17β-estradiol (37), arachidonic
acid
(AA)
(21),
epoxyeicosatrienoic
acids
(EETs)
(13),
and
dihydroxyeicosatrienoic acids (DHETs) (25) may be endogenous BK channel openers, and some transmitters and hormones can also 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 mainly mediated by the cannabinoid receptor, CB1, as shown by use of 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 cannabinoid receptor- and VR1-independent relaxation was sensitive to BK channel blockers, indicating that cannabinoids act on BK channels as “a potential endogenous 3
C-00482-2004 by Sade et al. R-2 BK channel opener” (30, 43). This fascinating hypothesis has been examined in a few studies (49, 33, and for review, 32), but not conclusively proven yet because of the multifunctional activity of AEA. For example, AEA directly blocks two pore-type K+ channels (TASK, 26) and T-type voltage-dependent Ca2+ channel currents (5), but activates VR1. The present study was undertaken to determine whether cannabinoids can activate BK channel currents expressed in human embryonic kidney 293 cells (HEK) in which the αsubunit of the BK channel (BKα), both α and β1 (BKαβ1), or both α and β4 (BKαβ4) subunits were heterologously expressed. We found that both AEA and a stable analogue, methanandamide (methAEA), have a potent BK channel–opening action.
4
C-00482-2004 by Sade et al. R-2 Methods Vector constructs, cell culture, and transfection. Restriction-enzyme–digested DNA fragments of BKα (Kpn I/Xba I-double digested), and BKβ1 and BKβ4 (EcoR I/ba I-double digested) were ligated into mammalian expression vectors, pcDNA3.1(+) and pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad, CA, USA), respectively, using the TaKaRa ligation kit Ver. 1 (TaKaRa) (45). Human embryonic kidney cell lines (HEK293) were obtained from the Health Science Research Resources Bank (HSRRB, Tokyo) and maintained in minimum essential medium (MEM, Gibco BRL, Rockville, MD, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS, JRS Biosciences, Lenexa, KS, USA), penicillin (100 units/ml, Wako, Osaka), and streptomycin (100 µg/ml, MEIJI SEIKA, Tokyo). Stable expression of BKα and BKβ was achieved by using calcium phosphate co-precipitation transfection techniques. Cells resistant to G418 (1 mg/ml, Gibco BRL) and G418/Zeocin (0.25 mg/ml, Invitrogen) were selected as those expressing BKα and co-expressing BKαβ, respectively. Expression of BKα and BKβ transcripts was confirmed by RT-PCR. Transfected cell lines were maintained in MEM medium supplemented with 10% FCS and G418 (0.5 mg/ml). The expression levels of BKα (~ 90%), BKαβ1 (~ 80%), and BKαβ4 (~80%) were confirmed using whole-cell or inside-out patch-clamp recordings of BK channel currents.
Cell dispersion. Male mice (Balb/c) weighing 20–30 g were anesthetized with ether and killed by exsanguination. After opening the chest, we excised an approximately 1.5 cm-long segment of 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 approx. 0.7 cm long in nominally Ca2+- and Mg2+-free Hanks solution for 5 min; the strips were then placed in solution containing 2 mg/ml collagenase (Amano, Nagoya, Japan) and 1mg/ml papain (Sigma) for 45 min. Following this step, the enzyme-treated strips were mechanically agitated in fresh Ca2+- and Mg2+-free Hanks solution that did not contain digestion enzymes. Dissociated cells were used within 6 h after cell dispersion. Ca2+- and Mg2+-free Hanks solution for cell-dispersion contained (mM) NaCl 137, KCl 5.4, Na2HPO4 0.168, KH2PO4 5
C-00482-2004 by Sade et al. R-2 0.44, glucose 5.55, and NaHCO3 4.17 (pH 7.45).
Solutions. The HEPES-buffered solution for electrophysiological recording had an ionic composition of (mM) NaCl 137, KCl 5.9, CaCl2 2.2, MgCl2 1.2, glucose 14, and HEPES 10. The pH of the solution was adjusted to 7.4 with NaOH. The pipette solution contained (mM) KCl 140, MgCl2 1, HEPES 10, Na2ATP 2, and EGTA 5. The pCa and pH of the pipette solution were adjusted to 6.5 and 7.2 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 (mM) KCl 140, MgCl2 1.2, glucose 14, HEPES 10, and EGTA 5. Selected pCa of the bathing solution was obtained by adding an adequate amount of CaCl2, and the 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 and aortic cells using a CEZ-2400 amplifier (Nihon Kohden, Tokyo). The procedures for electrophysiological recordings and data acquisition/analysis for whole-cell recording have been described previously (18). Whole-cell currents were recorded from each single 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 under the cell-attached or inside-out patch configuration and were analyzed by using the software, PAT V7.0C, developed at the University of Strathclyde (Dr. J. Dempster). The relative open-state probability of channels (NPo) was calculated using the following equation: N NPo = Σ (i * ti)/T i=0 , where “i” is the number of channels open, ti is the time spent “i” channels open, N is the maximum number of open channels observed in the patch, and T is the sampling time. In the 6
C-00482-2004 by Sade et al. R-2 present study, single-channel data for analysis were sampled for 60 s after the channel activity was stable. We did not examine the effect of cannabinoids on bursting behavior of BK channels, and records including long continuous closure of the channel (>10 s) were excluded. Because we did not define the total number of channels present in the cell-attached patch membrane, we assumed the maximum number of unitary current levels observed in a patch to be equal to the number of active channels in the patch. The single-channel data were sampled into a computer using the PAT program. Single-channel events were detected using a half-amplitude criterion, and the all-point amplitude histogram was fitted with the Gaussian distribution function. The resistance of the pipette was 2 to 5 MΩ for whole-cell and 10 to 15 MΩ for cell-attached patch and inside-out patch configurations when filled with the pipette solutions. The series resistance was partly compensated electrically under whole-cell voltage clamp. Whole-cell and single-channel recordings were carried out at room temperature (24±1 °C).
Ca2+ fluorescence measurements by Fura-2. The measurement of changes in cellular Ca2+ concentration ([Ca2+]i) by Fura-2 (Molecular Probes. Inc., Eugene, OR, USA) was performed as described previously (45). Prior to the fluorescence measurements, cells were incubated with 10 µM Fura-2AM in HEPES buffered solution for 30 min at room temperature. The fluorescence emission was collected from cell clusters using a dicroic 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: anandamide (AEA), methanandamide (methAEA), and penitrem A from Sigma-Aldrich (St. Louis, MO, USA); methAEA and cholera toxin (CTX) from Calbiochem; pertussis toxin (PTX) and PD98059 from Funakoshi (Tokyo, Japan); W-7 from Biomol. Res. Lab.; AM251 and JWH133 from TOCRIS; and KT5823 from Wako (Tokyo, Japan). PD98059, AM251, JWH133, and KT5823 7
C-00482-2004 by Sade et al. R-2 were dissolved in 100% DMSO and AEA and methAEA in 100% ethanol to make 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 mean±SEM in the text. Statistical significance between two groups and among multiple groups was evaluated using Student’s t-test and ANOVA following multiple comparisons, respectively. Symbols * and ** indicate P < 0.05 and P < 0.01, respectively.
8
C-00482-2004 by Sade et al. R-2 Results Effects of cannabinoids on macroscopic BK channel currents The effects of cannabinoids on BK channel currents were examined in single HEKBK αβ1 under whole-cell voltage clamp conditions. The Ca2+ concentration 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 and HEKBKαβ1, but the current density was much higher in the latter (the current density at the peak was 10.92±0.57 pA/pF for HEK and 20.73±2.0 for HEKBKαβ1; n=5 and 6, respectively; P0.05). These results strongly suggest that G proteins, indicating Gs and Gi, which have been shown to modulate BK channel activity in previous studies, are not direct targets of methAEA. It was also unlikely that calmodulin, MAP kinases, or G kinase mediated the methAEA-induced potentiation of BK channel currents because the potentiation was not significantly different from that observed following the pretreatment with 30 µM W7 (0.48±0.17 nA and 1.4±0.35 nA in the absence and presence of methAEA, respectively); 50 µM PD98059 (0.43±0.07 nA and 1.17±0.06 nA in the absence and presence of methAEA, respectively); or 0.3 µM KT5823 for 30 min (0.49±0.07 nA 1.26±0.14 nA in the absence and presence of methAEA, respectively) (Fig. 7B). [Fig. 7, here]
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+ concentration 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 13
C-00482-2004 by Sade et al. R-2 (228.8±41.6 pA and 510.0±77.5 pA in the absence an presence of methAEA, respectively; P
