Abstract
In this study, we focused on the pharmacological characterization of cannabinoid receptor coupling to G protein-gated inwardly rectifying potassium (GIRK) channels. Cannabinoids were tested on Xenopus laevis oocytes coexpressing the CB1 receptor and GIRK1 and GIRK4 channels (CB1/GIRK1/4) or the CB2 receptor and GIRK1/4 channels (CB2/GIRK1/4). WIN 55,212-2 enhanced currents carried by GIRK channels in the CB1/GIRK1/4 and CB2/GIRK1/4 system; however, the CB2 receptor did not couple efficiently to GIRK1/4 channels. In the CB1/GIRK1/4 system, WIN 55,212-2 was the most efficacious compound tested. CP 55,940 and anandamide acted as partial agonists. The rank order of potency was CP 55,940 > WIN 55,212-2 = anandamide. The CB1-selective antagonist SR141716A alone acted as a inverse agonist by inhibiting GIRK currents in oocytes expressing CB1/GIRK1/4, suggesting the CB1receptor is constitutively activated. A conserved aspartate residue, which was previously shown to be critical for G protein coupling in cannabinoid receptors, was mutated (to asparagine, D163N) and analyzed. Oocytes coexpressing CB1/GIRK1/4 or D163N/GIRK1/4 were compared. The potency of WIN 55,212-2 at the mutant receptor was similar to wild type, but its efficacy was substantially reduced. CP 55,940 did not elicit currents in oocytes expressing D163N/GIRK1/4. In summary, it appears the CB1 and CB2 receptors couple differently to GIRK1/4 channels. In the CB1/GIRK1/4 system, cannabinoids evaluated demonstrated the ability to enhance or inhibit GIRK currents. Furthermore, a conserved aspartate residue in the CB1 receptor is required for normal communication with GIRK channels in oocytes demonstrating the interaction between receptor and channels is G protein dependent.
The cannabinoid Δ-9-tetrahydrocannabinol (Δ9-THC) is the principal active constituent of marijuana (Gaoni and Mechoulam, 1964). It produces a multiplicity of central and peripheral effects, including euphoria, alteration in memory, analgesia, appetite stimulation, and immunomodulation. These effects are thought to be primarily the result of interactions with G protein-coupled receptors (GPCRs) denoted CB1 and CB2(Matsuda et al., 1990; Munro et al., 1993). There are many structurally diverse compounds that act as cannabinoid receptor agonists. The bicyclic analog of Δ9-THC, CP 55,940 [(−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol], and the structurally dissimilar aminoalkylindole, WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl) methyl]pyrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone], are agonists commonly used to test for cannabimimetic activity. Several endogenous ligands have also been proposed, including anandamide, an arachidonic acid derivative (Devane et al., 1992).
The CB1 receptor is located primarily in the brain (Herkenham et al., 1991), whereas the CB2receptor has been predominantly localized to cells of the immune system (Galiegue et al., 1995). With such diverse pharmacological effects produced from two receptors, it is perhaps not surprising that these receptors have been shown to couple to multiple effector systems. Activation of the CB1 receptor has been shown to affect cAMP levels, voltage-gated calcium channels, mitogen-activated protein kinase activity, and potassium channels (for a review, seePertwee, 1997). Unlike the CB1 receptor, activation of the CB2 receptor has been shown to alter only cAMP levels and mitogen-activated protein kinase activity. (Bouaboula et al., 1996).
G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in setting the membrane resting potential and influencing cell excitability (Hille, 1992). In the heart, the K(acetylcholine) channel is a GIRK1/GIRK4 (GIRK1/4) heterotetramer (Duprat et al., 1995; Krapivinsky et al., 1995). Binding of acetylcholine to cardiac muscarinic m2 receptors activates GIRK1/4 channels through G proteins and leads to the classic parasympathetic effect (i.e., decrease in heart rate; Pfaffinger et al., 1985). GIRK channels are also present in many brain regions and have been shown to couple to a variety of inhibitory neurotransmitter receptors, including opioid, muscarinic, and somatostatin receptors (Bausch et al., 1995;Chan et al., 1996; Hans-Jurgen et al., 1997). The physiological relevance of the interaction between receptor and channel protein in the brain is still unclear (for a review, see Dascal, 1997). Because the Gβγ subunits of G proteins are direct activators of GIRK, these channels allow the study of Gβγ effector outcomes (Logothetis et al., 1987; Reuveny and Jan, 1994).
The CB1 receptor activates GIRK channels in heterologous expression systems. Mackie et al. (1995) demonstrated in AtT-20 cells transfected with CB1 cDNA that the cannabinoid agonist WIN 55,212-2 enhanced current flux through an endogenous, pertussis toxin-sensitive, inwardly rectifying potassium channel. Similar results were obtained by Henry and Chavkin (1995) inXenopus laevis oocytes injected with the CB1 receptor and GIRK1 channel cRNA. On application of the cannabinoid agonist WIN 55,212-2, GIRK1 channel currents were enhanced. In a recent report describing the immunohistochemical distribution of the CB1receptor in rat brain, positively stained cap-like structures in the cerebellum were suggested to be basket cell projections. It was noted that these distinct structures appeared similar to projections that had been labeled positive with GIRK channel antibody in rat cerebellum (Tsou et al., 1998).
The aims of this investigation were to pharmacologically characterize CB1 receptor effects and to examine possible CB2 receptor coupling to GIRK channels. Previously, only a single cannabinoid agonist, WIN 55,212-2, had been evaluated for cannabinoid receptor activation of GIRK channels (Henry and Chavkin, 1995; Mackie and Mitchell, 1995). In the present study, multiple cannabinoid agonists were evaluated in a X. laevisoocyte system expressing the CB1 receptor and GIRK1/4 channel proteins (CB1/GIRK1/4). GIRK1/4 channels were used instead of GIRK1 alone because expression of GIRK channels as heterotetramers led to more consistent expression and increased activity (Duprat et al. 1995; Chan et al., 1996). The CB1-selective antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride], which has been reported to have inverse agonist activity (Compton et al., 1996; Bouaboula et al., 1997; Landsman et al., 1997), was also investigated to determine how it would affect the interaction between the CB1 receptor and GIRK1/4 channels. Finally, a CB1 receptor aspartate mutation, which has been previously shown to disrupt G protein coupling (Tao and Abood, 1998), was further characterized and used to demonstrate cannabinoid receptor communication with GIRK1/4 channels is dependent on G protein activation.
Experimental Procedures
Materials.
X.laevis frogs were purchased from Xenopus One (Dexter, MI). Reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). CP 55,940 was generously provided by Pfizer (Groton, CT). WIN 55,212-2 was purchased from Research Biochemicals Inc. (Natick, MA). Δ9-THC and SR141716A were obtained from NIDA. Dr. Raj Razdan (Organix, Woburn, MA) supplied the anandamide. The human CB1 cDNA was obtained from Dr. Marc Parmentier (Universite Libre de Bruxelles, Brussels, Belgium). The human CB2 cDNA was obtained from Dr. Sean Munro (Medical Research Council, Cambridge, UK). The rat GIRK1 cDNA was a gift from Dr. Henry Lester (California Institute of Technology, Pasadena, CA), and the cDNAs for the human homologs of GIRK1 and GIRK4, in the vector pGEM-HE, were graciously supplied by Dr. Diomedes E. Logothetis (Mt. Sinai Medical Center, New York, NY).
Mutagenesis.
The Altered Sites (Promega, Madison, WI) in vitro mutagenesis system was used to mutate the CB1receptor as previously described by Tao and Abood (1998). To make the D163N mutation, the mutagenic oligonucleotide (5′-CGGTGGCAAACCTCCTGGGGA) containing the desired mutation (GAC to AAC) was used. The mutations were confirmed by sequencing, and the mutated cDNA was subcloned into the mammalian expression vector pcDNA3 (InVitrogen, Carlsbad, CA).
cRNA Synthesis.
For preliminary studies, cDNAs for rat GIRK1 in pBluescript II KS−, human CB1 in pcDNA3, and human CB2 in pBluescript II KS− or in pGEM-HE were linearized by XhoI, XbaI,NotI, and NheI, respectively. For the heteromultimer studies, pGEM-HE containing the cDNAs for the human homologs of GIRK1 and GIRK4 were linearized using NheI. The cRNAs were transcribed in vitro using a T7 RNA polymerase (mMESSAGE mMACHINE; Ambion, Austin, TX).
Expression in Oocytes and Recordings.
Adult oocyte positiveX.laevis frogs were housed in distilled, dechlorinated water (18–20°C) with 12/12-h light/dark lighting cycle and fed twice weekly. Frogs were anesthetized by partial immersion in a 0.25% solution of MS-222 (Sigma Chemical Co., St. Louis, MO) for 10 to 30 min. A portion of the eggs was removed. The eggs were defolliculated with 1 mg/ml Collagenase type 1A (Sigma Chemical Co.) for 60 to 90 min. Rat GIRK1 or human GIRK1/4 and human CB1 or human CB2 cRNAs were coinjected into each oocyte (Drummond Scientific Co., Broomall, PA). Recordings were performed after 7 to 9 days of incubation in 0.5× L-15 media (Sigma Chemical Co., St. Louis, MO) supplemented with l-glutamine and antibiotics. For initial recordings, the eggs were placed in a chamber (total volume, 200 μl) and perfused at 4 ml/min with low potassium (LK) solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES pH 7.5), high potassium (HK) solution (2 mM NaCl, 96 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES pH 7.5), or HK plus drug. Fatty acid-free BSA (3 μM) was added to all drug solutions to minimize adsorption of cannabinoid compounds to the perfusion system. Oocytes were impaled with two microelectrodes filled with 3 M KCl (0.5–1.0 MΩ) and were voltage-clamped at reported voltages using an Axon GeneClamp amplifier (Axon Instruments Inc., Foster City, CA). Currents were collected at 100 Hz, filtered at 10 Hz, and analyzed using a Macintosh Centris 650 containing a 16-bit analog-digital interface board and voltage-clamp software running under the IGOR graphics environment (Wavemetrics, Lake Oswego, OR). Exchange of solutions was accomplished using a manual 6-port stream selection valve. In studies used to determine the KBvalue for the CB1-selective antagonist SR141716A, oocytes were pretreated with SR141716A for 5 min before the addition of WIN 55,212-2. When IGIRK amplitudes exceeded 2 μA, small transient changes in the IGIRK time course were often observed. These transient inward changes appeared within the first 20 s on application of drug. Extensive control studies revealed these inflections were not due to drug and/or any component of the vehicle. We concluded these inflections were due to an artifact of the solution exchange system that is more apparent at larger IGIRK amplitudes.
Data Analysis.
The EC50 andEmax values with corresponding 95% CLs were calculated using the Prism 2.0 program (GraphPAD, San Diego, CA). The Student’s t test (P < .05) was used for statistical analysis. The apparent equilibrium dissociation constant (KB) for the interaction of the antagonist and the receptor was calculated from the equationKB = [B]/(dose ratio − 1), where [B] is the concentration of antagonist used in the experiment (Griffin et al., 1998).
Results
Characteristics of CB1 and CB2 Receptors Coexpressed with GIRK Channels.
The addition of WIN 55,212-2 activated an inwardly rectifying potassium current when GIRK1 or GIRK1/4 cRNA were coinjected with human CB1 cRNA (Fig.1, A and B). In initial studies, oocytes were voltage-clamped at −80 mV and superfused in an LK solution containing 96 mM Na+ and 2 mM K+. When an HK solution containing 96 mM K+ and 2 mM Na+ was exchanged for LK, an inward current (IHK) was produced (Fig. 1B). IHK was a product of basal activation of GIRK1/4 channels (Chan et al., 1996). The application of the cannabinoid agonist WIN 55,212-2 in HK further enhanced the inward current and was defined as IAg. Coexpression of CB1 and GIRK1 subunits resulted in production of modest inward currents (Fig. 1A). The average IHK and IAg (in the presence of 1 μM WIN 55,212-2) were 772 ± 169 nA (n = 3) and 773 ± 78 nA (n = 3), respectively. In contrast, coexpression of CB1 and GIRK1/4 as a heteromultimers led to the production of robust inward currents. The average IHK and IAg (in the presence of 1 μM WIN 55,212-2) were 6.4 ± 1.1 μA (n = 5) and 4.7 ± 1.5 μA (n = 5), respectively (Fig.1B). In six different batches of oocytes injected with CB1/GIRK1/4, functional coupling between GIRK1/4 and the CB1 receptor, as defined by a response to agonist, was more than 96% (n = 48). In oocytes injected with CB1/GIRK1/4, Iag was also produced in the presence of the cannabinoid agonists anandamide and CP 55,940 (Fig. 1, C and D).
Oocytes coinjected with CB2 and GIRK1/4 cRNA demonstrated inconsistent coupling of less than 15% (n= 21), even when the amount of injected receptor cRNA was raised to 30 ng (injected per egg). In an effort to increase coupling efficiency, the CB2 receptor was placed into an vector specifically designed to increase expression of foreign cRNA translated proteins in X. laevis oocytes (Liman et al., 1992). In oocytes injected with 1 to 5 ng of cRNA, the CB2receptor coupled inconsistently, less than 20% (n = 43), to GIRK1/4 as was seen originally. When CB2cRNA concentrations of 11 to 34 ng were injected into the oocytes, cell integrity was compromised at day 3 (data not shown). We attempted to address the question of whether oocytes were expressing mature CB2 receptors using binding analysis but were unsuccessful because of high nonspecific binding in the oocyte membrane preparations. In oocytes responding to the cannabinoid agonist WIN 55,212-2, the average IHK and IAg were 4.8 ± 1.1 μA (n= 5) and 1.1 ± 0.6 μA (n = 5), respectively (Fig. 2A).
The currents produced by application of high potassium (IHK) are due to K+ flux through GIRK1/4 channels as well as rectifying channels endogenous to the oocyte (Kovoor et al., 1995). These currents can be separated using 300 μM Ba2+ (Dascal et al., 1993a), which blocks GIRK-mediated currents. To determine the fraction of native current (Inative) that was responsible for IHK, a pool of oocytes injected with only GIRK1/4 cRNA was tested for sensitivity to Ba2+ (Fig.2B). The average IHK was 6.6 ± 0.6 μA (n = 6). Inative (current not sensitive to 300 μM Ba2+) constituted only a small fraction of total IHK (443 ± 50 nA,n = 6). Inative was not significantly increased in the presence of 1 μM WIN 55,212-2 (837 ± 250 nA, n = 6). Currents carried by GIRK1/4 were not enhanced on application of WIN 55,212-2 in oocytes not injected with the CB1 receptor (Fig. 2B). Similar results were obtained in the presence of all cannabinoid agonists tested when the CB1 receptor was not coinjected (data not shown). Oocytes injected with the CB1receptor alone exhibited currents similar to Inative in the presence of HK and did not respond significantly to the application of 1 μM WIN 55,212-2 (Fig. 2C). Similar results were seen in oocytes injected with water (data not shown).
By definition, inwardly rectifying channels will display increasing inward current as the cell membrane is moved from positive to more negative potentials. To study whether cannabinoids can activate inward rectifiers, current-voltage plots were generated. Figure3 is a representative example of experiments in which oocytes injected with CB1/GIRK1/4 cRNA were subjected to voltage ramps (−80 to +50 mV) during treatment with HK or HK and 1 μM WIN 55,212-2 (WIN). Inward rectifying current activated under these conditions had a reversal potential near 0 mV, as expected for a potassium current because [K]i and [K]owere approximately equal. WIN 55,212-2 (1 μM) did not appear to shift the potassium equilibrium potential of the GIRK1/4 current but simply increased its amplitude. Therefore, current enhancement was most likely a result of increased GIRK1/4 channel conductance. Similar results were seen with all cannabinoid agonists tested (data not shown).
The initial recording protocol involved voltage clamping the oocytes at −80 mV. Under these conditions, IHK reached a short-lived steady state within the first 2 min, and then desensitization occurred for the remainder of the recording (Fig.4A). This slow agonist-independent desensitization has been reported to occur at the channel level (Kovoor et al., 1995). To carry out concentration-response experiments in a single oocyte, IHK was allowed to reach a stable steady state before application of the drug. Using this method, we observed compromised cell membrane integrity; therefore, the protocol was modified. Figure 4B is a representative example of typical concentration-response experiments using the modified recording approach. Oocytes injected with CB1/GIRK1/4 were voltage-clamped at 0 mV. In the presence of LK, HK, or HK plus increasing concentrations of WIN 55,212-2, 800-ms pulses of −80 mV were periodically applied to an oocyte to monitor the current (Fig.4B). This protocol allowed for IHK to reach a stable steady state (arrow) within approximately 17 min (n = 33). At this point, drug was added. Incomplete washout of drug is typically seen with cannabinoid compounds, presumably due to their lipophilicity; therefore, cumulative concentration-response experiments were performed to circumvent this problem. The application of increasing concentrations (4–5 concentrations per experiment) of WIN 55,212-2 led to a concentration-dependent enhancement of IAg (Fig.4C). A more detailed view of the IAg is shown in Fig. 4D. Figure 4B shows an example of the pulse recordings used to generate Fig. 4B. By following this recording protocol, stress to oocytes was minimal, allowing for increased data acquisition time and more reproducible concentration-response experiments.
Cannabinoid Receptor Agonists.
The responses to representative cannabinoid agonists CP 55,940, WIN 55,212-2, anandamide, and Δ9-THC were compared (Fig.5). These agonists produced a concentration-dependent enhancement of ionic current carried by GIRK1/4 channels in oocytes coinjected with CB1/GIRK1/4 cRNA. WIN 55,212-2 was chosen as the standard reference agonist and was run as a control in each set of experiments. A WIN 55,212-2 concentration-response curve was run each week, in each set of experiments, to standardize all agonist responses. The maximal response to WIN 55,212-2 was considered 100%. All responses were expressed as the percent of current enhancement: (IAg/maximal IAg produced with WIN 55,212-2) ∗ 100). Nonlinear regression analysis of WIN 55,212-2 gave EC50 andEmax values of 127 nM (64–251 nM) and 100% (81–118%), respectively. The potency of anandamide was not significantly different from WIN 55,212-2, but anandamide-stimulated currents were approximately half those seen with WIN 55,212-2. The EC50 and Emax values generated for anandamide were 89 nM (64–122 nM) and 46% (43–49%), respectively. CP 55,940 was the most potent compound tested, but it was the least efficacious (EC50 = 0.8 nM (0.2–3.2 nM) andEmax = 28% (19–37%), respectively). IAg was not consistently produced on application of Δ9-THC (data not shown); therefore, EC50 andEmax values could not be calculated.
Activity of SR141716A.
To determine whether the CB1-selective antagonist SR141716A (Rinaldi-Carmona et al., 1994) acts as an inverse agonist on GIRK1/4 channels, a concentration-response curve was generated. Substantial inhibition of IHK was produced by SR141716A in oocytes injected with CB1/GIRK1/4 cRNA but not in oocytes injected with only GIRK1/4 cRNA (Fig. 6). SR141716A inhibited IHK in a concentration-dependent manner (Fig.7A). The calculated EC50 andEmax values were 200 nM (100–300 nM) and −54% (48–61%), respectively. Figure 7B illustrates the magnitude of current inhibition produced by 1 μM SR141716A compared with 1 μM WIN 55,212-2. SR141716A or WIN 55,212-2 was applied to oocytes injected with CB1/GIRK1/4 cRNA when IHK reached steady state. The application of WIN 55,212-2 enhanced IHK by 104 ± 11% (n = 7), whereas SR141716A inhibited IHK by −46 ± 7% (n = 9).
In the next set of experiments, a concentration of SR141716A that did not produce significant inverse agonism (10 nM) was used to determine whether SR141716A could antagonize the effects of WIN 55,212-2 and anandamide (Fig. 8A). Concentrations that were the approximate EC80 values for the agonists were used in the presence of 10 nM SR141716A. WIN 55,212-2- and anandamide-stimulated currents were substantially reduced from 64 ± 4 to 25 ± 5% and 42 ± 5 to 27 ± 4%, respectively. In both the presence and absence of 10 nM SR141716A, two concentrations of WIN 55,212-2 located in the linear portion of the respective concentration-response curves were compared to calculate a dose ratio of 11.98 (7.82–18.57; Fig. 8B). The apparent equilibrium dissociation constant (KB) of SR141716A calculated in the presence of WIN 55,212-2 was 0.91 nM (0.57–1.46 nM).
Evaluation of Cannabinoid Receptor Mutant D163N.
Mutation of a conserved aspartate to an asparagine in the second transmembrane domain of the CB1 receptor disrupts G protein coupling (Tao and Abood, 1998). To compare the wild-type CB1 receptor with the mutant D163N receptor, concentration-response curves were generated with WIN 55,212-2 and CP 55,940 in oocytes expressing CB1/GIRK1/4 or D163N/GIRK1/4 (Fig. 9). Wild-type controls were compared with mutants in the same pool of oocytes. The potencies of WIN 55,212-2 and CP 55,940 in the wild-type (CB1/GIRK1/4) system were EC50 values of 103 nM (80–127 nM) and 1.3 nM (0.4–4.4 nM), respectively. Agonist-stimulated currents in the presence of WIN 55,212-2 and CP 55,940 in the wild-type system were Emax values of 103% (80–127%) and 27% (21–34%), respectively. The potency of WIN 55,212-2 was similar in the mutant (D163N/GIRK1/4) system, but agonist-stimulated currents in the presence of WIN 55,212-2 were substantially reduced. The calculated EC50 and Emaxvalues were 155 nM (76–316 nM) and 21% (18–24%), respectively.Emax and EC50 values could not be calculated for CP 55,940 in the mutant system because significant current enhancement was not seen.
Discussion
The results of this study demonstrate that a variety of cannabinoid agonists can enhance the activity of GIRK1/4 channels in oocytes coexpressing CB1/GIRK1/4. The effects produced by agonists were dependent on expression of the cannabinoid receptor. The current enhancement produced on application of WIN 55,212-2 was rapidly blocked by barium, which is known to block GIRK channel responses (Kovoor et al., 1995). In oocytes not injected with CB1/GIRK1/4, only native channel activity was observed in the presence of WIN 55,212-2. These results together suggest that the majority of current enhancement observed on application of cannabinoid agonists is due to the activation of GIRK1/4 channels. The current-voltage relationships generated in the presence and absence of cannabinoid agonists revealed that current enhancement is a result of increased GIRK1/4 channel activity.
There are several differences between the CB1 and CB2 receptors, including structure, tissue localization, ligand specificity, and, undoubtedly, function (Pertwee, 1997). The present investigation revealed the CB2receptor did couple to GIRK1/4 channels but not consistently. In addition, the agonist-stimulated currents, produced by WIN 55,212-2 in oocytes expressing CB2/GIRK1/4, were approximately four times less than those recorded in oocytes expressing CB1/GIRK1/4. A detailed dose-response analysis was not carried out in oocytes expressing CB2/GIRK1/4 because of the inconsistent coupling seen between receptor and channel. Perhaps the pertussis toxin-sensitive G proteins the CB2 receptor normally uses (Bouaboula et al., 1996) are only expressed at low levels in oocytes, and therefore activation of this pool of G proteins is inconsistent. However, it is more likely the receptor coupled promiscuously to G proteins it does not normally associate with in a native environment. The CB2 receptor has been shown not to couple to GIRK or calcium channels in AtT-20 cells (Mackie and Mitchell, 1995). Additionally, there is little evidence to suggest colocalization of the CB2 receptor and GIRK1/4 channels. CB2 receptors are restricted primarily to cells of the immune system, a population of cells not associated with expression of GIRK channels (Dascal et al., 1993b; Munro et al., 1993). In contrast, the CB1 receptor is located in many of the same regions of the central nervous system as GIRK channels (Herkenham et al., 1991; Ponce et al., 1996). CB1 receptors and GIRK channels could be colocalized on the action of γ-aminobutyric acid-positive neurons in the cerebellum (Tsou et al., 1998). In the brain, some of the effects of inhibitory neurotransmitter receptors are probably the result of the activation of GIRK channels (Dascal, 1997).
Whether CB1 receptors interact with GIRK channels in native tissue has yet to be determined. In this study, the EC50 values calculated for the cannabinoid agonists are in reasonable agreement with potency values reported in other functional assays, such as cannabinoid inhibition of forskolin-stimulated cAMP accumulation and cannabinoid stimulation of guanosine-5′-O-(3-thio)triphosphate binding (Felder et al., 1995; Griffin et al., 1998). WIN 55,212-2 was the most efficacious agonist tested in oocytes expressing CB1/GIRK1/4. In studies evaluating inhibition of calcium channels and neurotransmitter release, WIN 55,212-2 was reported to be the most efficacious compound tested (Pan et al., 1996; Shen et al., 1996). In this investigation, anandamide produced approximately 46% of the current enhancement seen with WIN 55,212-2, whereas CP 55,940 produced only 28% of the effect seen with WIN 55,212-2. The efficacy values reported for cannabinoid receptor inhibition of presynaptic glutamate release in rat hippocampal cultures (Shen et al., 1996) were similar to the efficacy values calculated in this study. It was suggested that ion channel modulation was the mechanism contributing to inhibition of glutamate release. The relationship between potencies in stimulation of GIRK channels and other pharmacological measures at least supports the premise that CB1 receptors may be coupled to GIRK channels.
Δ9-THC was tested in oocytes expressing CB1/GIRK1/4, but the production of concentration-dependent current enhancement was inconsistent, whereas WIN 55,212-2, CP 55,940, and anandamide were consistently effective as agonists. In other in vitro studies, Δ9-THC has acted as a weak partial agonist or has not produced an effect (Pertwee, 1997; Griffin et al., 1998). Receptor/G protein stoichiometry probably contributes to differences in the effects of the same compound between cell systems (for a review, see Kenakin, 1996).
The CB1-selective antagonist SR141716A (Rinaldi-Carmona et al., 1994) blocked the effects of WIN 55,212-2 and anandamide in oocytes injected with CB1/GIRK1/4 cRNA. The apparent equilibrium dissociation constant of SR141716A calculated in the presence of WIN 55,212-2 was similar to that reported for antagonism of agonist stimulated guanosine-5′-O-(3-thio)triphosphate binding in cerebellar membranes and agonist-induced inhibition of smooth muscle contraction (Pertwee and Griffin, 1995; Griffin et al., 1998). Although blockade of anandamide effects by SR141716A has previously been shown (Rinaldi-Carmona et al., 1994; Felder et al., 1995), a recent study demonstrated that SR141716A was not able to block a number of behavioral effects produced by anandamide in an established cannabinoid in vivo model, the mouse tetrad bioassay (Compton et al., 1993; Adams et al., 1998). Adams et al. (1998) suggested that metabolism may play a role in the effects of anandamide, albeit even anandamide metabolites acting at the CB1 receptor should be sensitive to SR141716A blockade. In the present study, a single oocyte is constantly perfused (4 ml/min) with anandamide; therefore, effects produced by anandamide metabolites are unlikely. Thus, in oocytes expressing CB1/GIRK1/4, anandamide enhances GIRK currents through a CB1 receptor-mediated pathway that is sensitive to blockade by SR141716A.
It was quite evident that SR141716A was an effective antagonist when evaluated at concentrations (10 nM) that were devoid of any direct effects. However, the application of SR141716A alone at concentrations greater than 10 nM inhibited GIRK currents. This latter effect was dependent on expression of the CB1 receptor and demonstrates that a single GPCR subtype can both stimulate and inhibit the activity of GIRK1/4 channels. There is increasing evidence that SR141716A can behave as a inverse agonist in vivo and in vitro (Compton et al., 1996; Bouaboula et al., 1997; Landsman et al., 1997). If the CB1 receptor can affect GIRK channels in native tissue, then perhaps some of the inverse agonist activity produced by SR141716A in vivo is related to this interaction.
A recent report by Pan et al. (1998) proposed that the inverse agonist qualities of SR141716A are a result of inhibition of constitutively active CB1 receptors. In X. laevisoocytes, even in the absence of expressed GPCRs, GIRK1/4 channels have a substantial basal activity that is G protein dependent (Chan et al., 1996). If constitutively activated CB1 receptors were shifted into an inactive state by SR141716A, the signaling moieties responsible for maintaining basal activity of GIRK channels (i.e., free Gβγ subunits) could be sequestered by the inactivated CB1 receptors. The result would be a decrease in the basal activity of the GIRK channels. Alternatively, if an oocyte could synthesize a sufficient amount of an endogenous agonist, such as anandamide, to activate CB1 receptors, then a neutral antagonist could appear to be acting as a inverse agonist. However, it is highly unlikely that a single oocyte, which is rapidly perfused, could continually produce a significant concentration of an endogenous agonist.
The use of pertussis toxin in oocytes to suggest an effect is Gi/Go protein dependent can lead to conflicting results because oocytes can contain pertussin toxin-insensitive forms of Gi/Go (Dascal et al., 1993a). We previously reported that mutation of the CB1 receptor to D163N resulted in a receptor that retained binding affinity for cannabinoid ligands but lost the ability to fully couple to G proteins (Tao and Abood, 1998). Specifically, the D163N receptor could only partially inhibit cAMP accumulation even when high doses of cannabinoid agonists were used. An analogous mutation in the α2-adrenoreceptor resulted in a receptor that was selectively uncoupled from one of the two (cAMP and GIRK) second messenger systems studied (Surprenant et al., 1992).
The potency value for WIN 55,212-2 was not significantly different in oocytes expressing D163N/GIRK1/4 versus the wild-type system. However, current enhancement produced by WIN 55,212-2 was substantially reduced in the mutant system. A potency value for CP 55,940 could not be obtained because significant current enhancement was not produced by this ligand in the D163N/GIRK1/4 system. The results with WIN 55,212-2 suggest that the ligand was able to bind to the mutant receptor but that uncoupling of receptor and G proteins resulted in an substantial attenuation of current enhancement. Although CP 55,940 may have still bound to the mutant receptor, its lower efficacy compared with WIN 55,212-2 probably prevented it from producing a significant response. These interpretations would be in agreement with our previously published results. The results of the mutation experiments suggest the interaction between the CB1 receptor and GIRK1/4 channel is G protein dependent. In the CB1receptor, the aspartate residue is part of a critical structural requirement that allows efficient coupling to not only cAMP but also GIRK channels.
In summary, the results demonstrate the functional coupling of CB1 and CB2 receptors to GIRK1/4 channels, albeit the CB2 receptor couples less efficiently. Although previous studies have used only a single cannabinoid agonist, this investigation provides detailed pharmacological analysis in a system that demonstrated G protein-dependent coupling between cannabinoid receptors and GIRK channels. In oocytes expressing CB1/GIRK1/4, SR141716A can act as an antagonist at low concentrations. At higher concentrations, it acts as an inverse agonist, demonstrating that GIRK channels can be both inhibited and activated by a single GPCR subtype. Finally, it appears the aspartate residue in the second transmembrane of the CB1 receptor is critical for coupling to multiple effector systems. Future research into cannabinoid receptor second messenger systems and structure activity should provide us with insight into the role of the cannabinoid system in humans.
Acknowledgments
We thank Dr. Diomedes E. Logothetis for his suggestions on recording techniques for concentration-response analysis. We also thank Qing Tao for constructing the D163N mutant, Dr. Tooraj Mirshahi for his advice during the initial development of the CB1/GIRK1/4 system, and Drs. Tracie Kinard and Billy R. Martin for their critical input. Finally, we thank Tara Blevins and Dr. John Woodward for invaluable support of the oocyte facility.
Footnotes
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Send reprint requests to: Dr. Mary E. Abood, Forbes Norris ALS Research Center, 2351 Clay St., Suite 416, California Pacific Medical Center, San Francisco, CA 94115. E-mail:mabood{at}cooper.cpmc.org
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↵1 This work was supported by National Institutes of Health Grants DA09978 and DA05274 (to M.E.A.) and DA07027 and DA05910 (training support for S.D.M.).
- Abbreviations:
- Δ9-THC
- Δ-9-tetrahydrocannabinol
- CB1 and CB2
- cannabinoid receptor
- HP
- high potassium
- GIRK
- G protein-coupled inwardly rectifying potassium
- G protein
- guanine nucleotide binding protein
- GPCR
- G protein-coupled receptor
- IHK
- inward current with an HK solution containing 96 mM K+ and 2 mM Na+ exchanged for LK
- IAg
- inward current with application of the cannabinoid agonist WIN 55,212-2 in HK
- Inative
- native current
- CP55,940
- (−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol
- WIN 55,212-2
- (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone
- SR141716A
- N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride
- Received May 5, 1999.
- Accepted July 22, 1999.
- The American Society for Pharmacology and Experimental Therapeutics