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436

Effects of cannabinoids on endogenous K+ and

Ca2+ currents in HEK293 cells
Clemente Vásquez, Ricardo A. Navarro-Polanco, Miguel Huerta, Xóchitl Trujillo,
Felipa Andrade, Benjamín Trujillo-Hernández, and Leonardo Hernández

Abstract: Effects of cannabinoids on endogenous potassium and calcium currents in HEK293 cells were studied using
the whole-cell variant of the patch-clamp technique. The cannabinoid agonists WIN 55,212-2, methanandamide, and
anandamide (1 µM) decreased the calcium current by 53.1 ± 2.6, 47.5 ± 1.2, and 38.8 ± 3.1%, respectively, after
transfection of human CB1 cannabinoid receptor (hCB1) cDNA into HEK293 cells. The delayed rectifier-like current
was not changed after application of these agonists, but the inward rectifier was increased by 94.0 ± 3.6, 83.7 ± 5.1,
and 63.0 ± 2.5% after application of WIN 55,212-2, methanandamide, and anandamide, respectively. The effects of the
cannabinoid antagonists (AM251, AM281, and AM630) on the inward rectifier and calcium currents were the opposite
of those seen with cannabinoid agonists; thus, these compounds act as inverse agonists in this preparation. These re-
sults suggest that endogenous inward rectifier and calcium currents are modulated by cannabinoids in HEK293 cells,
and that some expressed receptors may be constitutively active.
Key words: cannabinoids, WIN 55,212-2, anandamide, methanandamide, inverse agonists.
Résumé : On a examiné les effets des cannabinoïdes sur les courants potassiques et calciques endogènes dans les cel-
lules HEK293 en utilisant la technique du patch clamp en configuration cellule entière. Les agonistes cannabinoïdes,
WIN 55,212-2, méthanandamide et anandamine (1 µM) ont diminué le courant calcique de 53,1 ± 2,6, 47,5 ± 1,2 et
38,8 ± 3,1 %, respectivement, après la transfection de l’ADNc du récepteur cannabinoïde CB1 humain (CB1h) dans les
cellules HEK293. Le courant redresseur tardif n’a pas été modifié après l’application de ces agonistes, mais le courant
redresseur entrant a été augmenté de 94,0 ± 3,6, 83,7 ± 5,1 et 63,0 ± 2,5 % après l’application de WIN 55,212-2, de
méthanandamide et d’anandamine, respectivement. Les effets des antagonistes cannabinoïdes (AM251, AM281 et
AM630) sur les courants calciques et redresseurs entrants ont été a l’opposé de ceux des agonistes cannabinoïdes;
ainsi, ces composés se comportent comme des agonistes inverses dans cette préparation. Ces résultats donnent à penser
que les courants calciques et redresseurs entrants endogènes sont modulés par les cannabinoïdes dans les cellules
HEK293 et que certains récepteurs exprimés pourraient être constitutivement actifs.
Mots clés : cannabinoïdes, WIN 55,212-2, anandamide, méthanandamine, agonistes inverses.
[Traduit par la Rédaction] Vásquez et al. 442

Introduction
The brain CB1 cannabinoid receptor is a member of the
G-protein-coupled receptor superfamily (Matsuda et al.
1990). Among the wide variety of effects due to activation
of CB1 cannabinoid receptors are the following: inhibition
of glutamatergic and GABAA synaptic transmission and re-
lease of GABA and acetylcholine from hippocampal neurons
Received 31 July 2001. Published on the NRC Research
Press Web site at http://cjpp.nrc.ca on 10 April 2003.
C. Vásquez,1 R.A. Navarro-Polanco, M. Huerta,
X. Trujillo, F. Andrade, and L. Hernández. Centro
Universitario de Investigaciones Biomédicas, Universidad de
Colima, Avenida 25 de julio # 965, Colonia Villa de San
Sebastián, Colima, Colima, C.P. 28040, México.
B. Trujillo-Hernández. Instituto Mexicano del Seguro Social
(I.M.S.S.), Unidad de Investigación en Epidemiología Clínica,
Hospital General de Zona y Medicina Familiar No. 1,
Avenida de los Maestros # 140, Colima, Colima, C.P. 28000,
México.
1
Corresponding author (e-mail: clemvas@cgic.ucol.mx).
(Gifford and Ashby 1996; Katona et al. 1999; Hoffman and
Lupica 2000), inhibition of dopamine release in rat brain
(Kathmann et al. 1999), and inhibition of serotonin release
in mouse brain (Nakazi et al. 2000). Thus, the CB1 canna-
binoid receptor modulates neuronal excitability and neuro-
transmitter release, and thus regulates Ca2+ and K+ currents
(Kim and Thayer 2000). Activation of CB1 receptors inhib-
its the N- and P-/Q-type Ca2+ channels in cultured hippo-
campal neurons and in heterologous expression systems
(Pan et al. 1996; Twitchell et al. 1997). On the other hand,
CB1 receptors activate inwardly rectifying K+ channels
(Mackie et al. 1995), while it was recently shown that
cannabinoids decrease the K+ M-current in hippocampal
CA1 neurons (Schweitzer 2000). As already is noted, sev-
eral systems have been used to observe the effects of canna-
binoids on ionic currents. Another cell line used extensively
for transfection experiments, HEK293 cells, has endogenous
K+ and Ca2+ currents (Berjukow et al. 1996; Yu and
Kerchner 1998), and we wondered whether these currents
undergo the same modulations by cannabinoids as those de-
scribed above. If so, we could have an additional tool with
which to study the mechanisms underlying the physiological

Can. J. Physiol. Pharmacol. 81: 436–442 (2003) doi: 10.1139/Y03-055 © 2003 NRC Canada

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Vásquez et al.
actions of cannabinoids and the endogenous ligands for
these receptors.
Materials and methods
Cell culture and transfection
HEK 293 cells were grown at 37°C in Dulbecco’s modi-
fied Eagle’s medium (GIBCO, Rockville, Md.) containing
10% horse serum under 5% CO2.
Human CB1 cannabinoid-receptor cDNA was inserted
into the mammalian expression vector pCI (Promega,
Madison, Wis.) and expressed under the control of a cyto-
megalovirus (CMV) promoter. HEK293 transfections were
mediated by Effectene transfection reagent (QIAGEN, Va-
lencia, Calif.), the manufacturer’s instructions being fol-
lowed exactly. A standard transfection (in 35-mm Petri
dishes) was carried out using 0.1 µg of each expression
plasmid, and 10 µL Effectene transfection reagent at a cell
density of about 70% confluency. The green fluorescent pro-
tein was used as a cotransfection marker to identify cells that
had been successfully transfected. Patch-clamp experiments
were performed 24–48 h after transfection.
Electrical recording
Potassium and strontium currents were recorded in
HEK293 cells at 22–24°C by means of the whole-cell vari-
ant of the patch-clamp technique (Hamill et al. 1981), a
patch-clamp amplifier (Axopatch 200B, Axon Instruments,
Foster City, Calif.) being used for this purpose. Strontium
currents were filtered at 1 kHz (four-pole Bessel filter) and
digitized at a sampling rate of 5 kHz. Potassium currents
were filtered at 3 kHz and digitally sampled at 10 kHz, and
leak currents were substracted.
Voltage-clamp protocols were generated by a Compaq
S500 computer (Pentium III) using the program pClamp 6.0
(Axon Instruments, Foster City, CA, U.S.A.). Data were dig-
itized using a DIGIDATA 1200 interface (Axon Instruments,
Foster City, CA, U.S.A.) and stored on the computer hard
disc. Depolarizing the cell membrane from a holding poten-
tial of –70 mV to above threshold (–40 mV) evoked tran-
sient voltage-dependent inward strontium currents
(Berjukow et al. 1996). K+ currents were elicited by voltage
steps from a holding potential of –70 mV. Depolarizing steps
to +40 mV from the holding potential triggered an outward
potassium current, while hyperpolarizing steps to –150 mV
from the holding potential activated a small inward potas-
sium current (Yu and Kerchner 1998).
Results are expressed as the mean ± SE. Figures were
generated using Clampfit (Axon Instruments) with final
preparation in Sigmaplot (SPSS Inc., Chicago, Ill.).
Solutions and drugs
To isolate strontium currents for whole-cell recording,
cells were bathed in an external solution containing 10 mM
SrCl2, 190 mM N-methyl-D-glucamine, 10 mM HEPES,
20 mM glucose, 4 mM 4-aminopyridine, 27 mM tetraethyl-
ammonium chloride, and 3 mM MgCl2, buffered to pH 7.3
with methanesulphonic acid. The intracellular solution for
recording strontium currents consisted of 60 mM CsCl,
60 mM CsOH, 60 mM aspartate, 2 mM MgCl2, 10 mM
HEPES, and 10 mM EGTA, titrated to pH 7.25 with CsOH.
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437
The external solution for recording the K+ currents con-
tained 110 mM NaCl, 2.5 mM KCl, 2.0 mM MnCl2, 0.1 mM
BAPTA, 10 mM HEPES, and 10 mM glucose. The internal
solution for recording K+ currents contained 120 mM KCl,
1.0 mM CaCl2, 1.5 mM MgCl2, 1.0 mM BAPTA, 10 mM
HEPES, and 2 mM Na-ATP. MnCl2 and TTX (0.1 µM) were
included in the external solution to block Ca2+ and Na+
channels, respectively. Each solution had a pH of 7.3–7.4.
Drugs solutions were applied to isolated cells by bath per-
fusion. The stock solutions of 10 mM WIN 55,212-2
mesylate, AM251, AM281, and AM630 (Tocris Cookson
Inc., Ballwin, Mo.) were made up in dimethylsulphoxide.
Stock solutions of 10 mM anandamide (Tocris Cookson
Inc.) and methanandamide (Research Biochemicals Interna-
tional, Natick, Mass.) were made up in ethanol. The final
concentrations of dimethylsulfoxide and ethanol were
<0.01%, which had no effect on the K+ and Ca2+ currents.
Results
Effects of cannabinoids on endogenous K+ currents in
HEK293 cells
The presence of at least two different endogenous potas-
sium currents has been described in HEK293 cells: (i) a de-
layed rectifier-like current and (ii) a small inward rectifier
current (Yu and Kerchner 1998). Figure 1 shows these cur-
rents as obtained in the present study and, as can be appreci-
ated, the currents are similar to those reported before in
nontransfected HEK293 cells (Yu and Kerchner 1998). De-
layed rectifier current was suppressed by TEA (20 mM) at
+40 mV by 55.9 ± 10.2%; when the external K+ concentra-
tion was increased from 2.5 to 25 mM, it attenuated the cur-
rent by 19 ± 3%; substituting Cs+ for K+ in the recording
pipette solution eliminated the current; delayed current
showed some sensitivity to 4-aminopyridine and the current
had outward rectification at depolarized potentials and lack
of inactivation during a 400-ms pulse (Yu and Kerchner
1998). Hyperpolarizing voltage pulses for triggering inward
rectifier current activated a small inward current larger at
more negative potentials; when most external Cl- was re-
placed by acetate, no effect on the outward current was ob-
served; these currents were recorded in the presence of Na+
and Ca2+ channel antagonist, thus, contributions from these
two types of voltage-gated channels could be excluded;
varying external Ca2+ concentration did not affect current
size; charybdotoxin (100 nM), apamin (1 µM), and
tolbutamine (10 µM) showed no effect on the current (Yu
and Kerchner 1998). In our study, these currents were ob-
served in HEK293 cells transfected with hCB1 cannabinoid-
receptors cDNA. In fact, such K+ currents were observed in
22 (73%) of 30 transfected HEK293 cells. After recording
the currents, we tested the effect of several cannabinoid
agonists on them. Application of 1 µM of WIN 55,212-2,
methanandamide, or anandamide increased the inward recti-
fier current (Fig. 1), the rank order of sensitivities being as
follows: WIN 55,212-2 > methanandamide > anandamide.
The dose used (1 µM) is within the range employed in other
heterologous systems to study the actions of similar drugs
(Pan et al. 1996, 1998; Vásquez and Lewis 1999).
The increases due to WIN 55,212-2, methanandamide,
and anandamide were by 94.0 ± 3.6 (n = 5), 83.7 ± 5.1 (n =
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438 Can. J. Physiol. Pharmacol. Vol. 81, 2003

Fig. 1. Effects of WIN 55,212-2 (WIN), methanandamide (METHAN), and anandamide (ANAND) on endogenous K+ currents in
HEK293 cells. (A) Superimposed traces of delayed rectifier current from a HEK293 cell in the absence (control) and presence of WIN
55,212-2 (10 µM WIN). Currents were induced by depolarizing voltage pulses from –70 mV to a test potential of +20–100 mV.
(B) Superimposed traces of inward rectifier current from the same cell in the absence (control) and presence of WIN 55,212-2 (1 µM
WIN). The HEK293 cell was hyperpolarized from a holding potential of –70 mV to a test potential of –150 mV. (C) Bar graph show-
ing the increment in the inward rectifier current induced by WIN 55,212-2, methanandamide, and anandamide in HEK293 cells
transfected with human cannabinoid receptor CB1 cDNA. Baseline of currents are indicated by dashed lines.

5), and 63.0 ± 2.5% (n = 4), respectively. These effects were
all reversible. Cannabinoid agonists had no effect in untrans-
fected cells (WIN 55,212-2: 2.1 ± 0.6%, n = 5; methanan-
damide: 1.5 ± 0.4%, n = 5; anandamide: 2.6 ± 0.9%, n = 4).
The delayed-rectifier-like current was not modified by
cannabinoid agonists, even at concentrations as high as
10 µM (Fig. 1).
The application of cannabinoid antagonists confirmed that
the action of the above agonists was mediated by CB1
cannabinoid receptors, because 1 µM of AM251, AM281, or
AM630 blocked the effects of the cannabinoid agonists (data
not shown). The dose used of canabinoid antagonists (1 µM)
is within the range employed in other heterologous systems
to study the actions of similar drugs (Pan et al. 1996, 1998;
Vásquez and Lewis 1999).
Interestingly, as well as blocking the action of the agonists,
the antagonists had effects by themselves on the potassium cur-
rent. In fact, they each produced a decrement in the inward rec-
tifier current (Fig. 2), AM251 reducing it by 47.3 ± 4.7% (n =
4), AM281 by 52.7 ± 2.1% (n = 4), and AM630 by 51.0 ±
6.0% (n = 5). In Fig. 2B, a current–voltage (I–V) relationship
is presented to show the effects of the cannabinoid agonist
WIN 55,212-2 and the cannabinoid antagonist AM630 on K+
current. As it is noted, drugs effects are opposite.
Effects of cannabinoids on endogenous Ca2+ currents in
HEK293 cells
At least one endogenous calcium current has been de-
scribed in HEK293 cells (Berjukow et al. 1996). The current
obtained in the present study is similar to the one reported be-
fore in nontransfected HEK293 cells (Berjukow et al. 1996),
and we observed it in HEK293 cells transfected with hCB1
cannabinoid-receptor cDNA, too. This Ca2+ current was ob-
served in 23 (77%) of 30 transfected HEK293 cells, with
10 mM strontium being used as the charge carrier. The cur-
rent activated on depolarization to voltages positive of –40
mV, reached maximal values at –6 ± 1.0 mV, and reversed at
55 ± 6.1 mV. Block of these currents by the dihydropyridine
isradipine was not stereoselective: 1 µM (+)- and (–)-
isradipine inhibited current by 30 ± 4% and 29 ± 2%, re-
spectively; 10 µM (–)-nimodipine inhibited the current by
78 ± 6%; the cone snail toxins ω-CTx GVIA and ω-CTx
MVIIC (1 µM) inhibited the current by 17 ± 3 and 20 ± 3%,
respectively; and the funnel web spider toxin ω-Aga IVA
(200 nM) inhibited the current by 19 ± 2% (Berjukow et al.
1996).
The cannabinoid agonists WIN 55,212-2, methanan-
damide, and anandamide (1 µM) decreased the calcium cur-
rent by 53.1 ± 2.6, 47.5 ± 1.2, and 38.8 ± 3.1%, respectively

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Fig. 2. Effects of cannabinoid antagonists on inward rectifier
current in HEK293 cells. (A) Superimposed traces of inward rec-
tifier current from another HEK293 cell (i.e., different cell from
that in Fig. 1) in the absence (control) and presence of AM630
(1 µM). Currents were induced by hyperpolarizing voltage pulses
from –70 mV to a test potential of –150 mV. (B) I–V relation-
ship showing effects of WIN 55,212-2 and AM630 on K+ cur-
rent. Both drugs were tested with a concentration of 1 µM.
Symbols represent the mean of 4–5 experiments. (C) Bar graph
showing inhibition of the inward rectifier current by AM251,
AM281, and AM630 in HEK293 cells transfected with human
cannabinoid receptor CB1 cDNA. Baseline of currents are indi-
cated by dashed lines.
(n = 4 for each agonist) in cells transfected with hCB1
cDNA (Fig. 3) but had no effect in untransfected cells (WIN
55,212-2: 1.1 ± 0.9%, n = 4; methanandamide: 2.5 ± 0.7%,
n = 5; anandamide: 1.6 ± 1.0%, n = 5).
To test whether hCB1 cannabinoid receptors are in an ac-
tive state in HEK293 cells transfected with hCB1, several
antagonists of these receptors were applied. AM251,
AM281, and AM630 (1 µM), in addition to blocking the ef-
fect of the above agonists, increased the calcium current by
30.2 ± 4.1, 34.6 ± 2.3, and 33.0 ± 4.5%, respectively (n = 3
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439
for each antagonist) when each was applied by itself
(Fig. 3). These antagonists had no effects on untransfected
cells (AM251: 0.8 ± 0.3%, n = 5; AM281: 2.8 ± 1.3%, n =
5; AM630: 1.2 ± 0.9%, n = 5). In Fig. 3E, an I–V relation-
ship is presented to illustrate the actions of the cannabinoid
agonist WIN 55,212-2 and cannabinoid antagonist AM630.
It can be appreciated that drugs effects are opposing.
These observations, together with the effects of canna-
binoid antagonists on the inward rectifier current, suggest
that some expressed cannabinoid receptors in HEK293 cells
are in a constitutively active state, as previously described in
other preparations (Bouaboula et al. 1997; Landsman et al.
1997; MacLennan et al. 1998; Pan et al. 1998; McAllister et
al. 1999).
Discussion
The main purpose of the present study was to characterize
the effect of cannabinoids on endogenous K+ and Ca2+ cur-
rents in HEK293 cells. In fact, we found that cannabinoids
activate an inward rectifier-like current and inhibit a Ca2+
current. In this sense, cannabinoids had similar effects in
HEK293 cells as in neurons expressing cannabinoid recep-
tors or in heterologus expression systems (Mackie et al.
1995; Pan et al. 1996; Twitchell et al. 1997). Thus, the cellu-
lar mechanism by which CB1 receptors modulate the endog-
enous channels in HEK293 cells would be by activation of
the pertussis-toxin-sensitive G-proteins pathway of the Gi
family (such as Gi or Go) (Mackie et al. 1995; Pan et al.
1996; Twitchell et al. 1997). Indeed, the presence of Gi pro-
teins in HEK293 cells has been described (Mundell and
Benovic 2000). Further work is needed to explore the spe-
cific type of subunits (α, β, and γ) of G proteins that medi-
ates this pathway.
The Ca2+ currents recorded in the present study are similar
that those published by Berjukow et al. (1996), as our I–V
curve shows a similar voltage dependence as that reported
by those authors. Endogenous Ca2+ channels in HEK293
cells share some properties with the low-voltage-activated
class-E calcium channel or T-type channel but may represent
a new class of voltage-dependent calcium channels. Thus,
we acknowledge the uncertainties of the molecular basis for
these currents, as have been recognized by others (Berjukow
et al. 1996); this could be one limitation of our study. Even
when the inward-current density is reportedly low (0.39 ±
0.7 pA/pF; Berjukow et al. (1996)), the effects of canna-
binoids can be observed. Inhibition of these Ca2+ channels
will decrease the likelihood of both neurotransmitter release
and successful synaptic transmission, as well as suppressing
other calcium-dependent processes.
Activation of the inward rectifier current would inhibit
cellular excitability, because the membrane would be held at
a negative potential with respect to the threshold for activa-
tion of Na+ and Ca2+ channels, thus producing alterations in
action-potential generation and consequently interrupting
cellular communication.
Among the endogenous cannabinoids tested, anandamide
had the weakest effect on K+ and Ca2+ currents. This was
also observed by Mackie et al. (1993) and by Gebremedhin
et al. (1999). However, Mackie et al. (1995) and Twitchell et
al. (1997) reported that the activity of anandamide was equal
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440 Can. J. Physiol. Pharmacol. Vol. 81, 2003

Fig. 3. Effects of cannabinoid agonists and antagonists on Ca2+ channel currents in HEK293 cells. (A) Superimposed traces of Ca2+-
channel currents from a HEK293 cell in the absence (control) and presence of WIN 55,212-2 (1 µM WIN). The HEK293 cell was de-
polarized from a holding potential of –70 mV to a test potential of 0 mV. (B) Summary of the inhibition of Ca2+ channel currents by
WIN 55,212-2 (WIN), methanandamide (METHAN), and anandamide (ANAND) in HEK293 cells transfected with human cannabinoid
receptor CB1 cDNA. (C) Superimposed traces of Ca2+ channel currents from a HEK293 cell in the absence (control) and presence of
AM630 (1 µM). Currents were induced by depolarizing voltage pulses from –70 mV to a test potential of 0 mV (D). Summary of the
increments in the Ca2+ channel currents induced by AM251, AM281, and AM630 in HEK293 cells transfected with human canna-
binoid receptor CB1 cDNA. (E) I–V relationship showing effects of WIN 55,212-2 and AM630 on Ca+2 current. Both drugs were
tested with a concentration of 1 µM. Symbols represent the mean of 4–5 experiments.

to that of WIN 55,212-2, while Pan et al. (1996) observed
inconsistent effects of anandamide in a heterologous expres-
sion system. These discrepancies could be explained by
there being different densities of CB1-receptor expression in
the different systems (Twitchell et al. 1997).
In the smooth muscle cells of the rat aorta, although WIN
55,212-2, anandamide, and methanandamide all inhibited the
delayed rectifier K+ current, and SR141716A neither pre-
vented the influence of these cannabinoid agonists nor had
any effect by itself (Van den Bossche and Vanheel 2000).
These findings suggest that the observed inhibitory action of
cannabinoids in that preparation does not depend on CB1-
receptor stimulation. Possibly, it is produced by an interac-
tion with another as yet undescribed, cannabinoid receptor.
Besides, recently, Chemin et al. (2001) demonstrated that
anandamide can directly block T-type Ca2+ channels in the
absence of CB1/CB2 receptors. Thus, is important for us to
know the way different preparations behave to be able to un-
derstand cannabinoid-receptor physiology completely. To
our knowledge, this is the first evidence showing modulation
of endogenous K+ and Ca2+ channels in HEK293 cells by
cannabinoids.
It was previously reported that SR141716A, a cannabinoid
antagonist, acts as an inverse agonist on hCB1 receptors.
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With binding studies, it was showed that guanine nucleo-
tides decreased the binding of the cannabinoid agonist CP-
55,940, whereas it enhanced the binding of SR141716A, a
property of inverse agonists (Bouaboula et al. 1997). The
basal incorporation of [35S]-GTPgammaS in cells express-
ing human recombinant CB1 receptors was inhibited by
SR141716A, whereas cannabinol (cannabinoid agonist) had
no significant effect (MacLennan et al. 1998). SR141716A
abolished the inhibition of Ca2+ currents by the agonist WIN
55,212-2 and increased Ca2+ currents in neurons micro-
injected with CB1 cRNA. For an antagonist to elicit an ef-
fect, some receptors must be tonically active, and it acts as
an inverse agonist (Pan et al. 1998); in oocytes coexpresing
CB1 receptors and G protein-gated inwardly rectifying po-
tassium channels, WIN 55,212-2 enhanced currents and
SR141716A alone, inhibited potassium currents (McAllister
et al. 1999). All these authors suggested that cannabinoid re-
ceptors are in a constitutively active state. When we tested
the other cannabinoid antagonists AM251, AM281, and
AM630, each of which has also been demonstrated to act as
an inverse agonist, AM630 produced a decrease in the maxi-
mal response of electrically evoked twitches of the mouse
isolated vas deferens apart of blocking effects of canna-
binoid agonist (Pertwee et al. 1995); AM281 robustly poten-
tiated electrically evoked release of acetylcholine in
hippocampal slices and prevented the inhibition of acetyl-
choline release by WIN55,212-2 (Gifford et al. 1997);
AM630 inhibited [35S]GTP γ S binding to cannabinoid recep-
tor membranes, enhanced forskolin-stimulated cyclic AMP
production, and antagonized the inhibition of forskolin-
stimulated cyclic AMP production induced by cannabinoid
agonist in transfected cells (Ross et al. 1999); we found
AM251, AM281, and AM630 all to act as inverse agonists
in HEK293 cells expressing hCB1 cannabinoid receptors,
since by themselves they had effects on ionic currents.
These effects were opposite to the effects of the cannabinoid
agonists. This is the first demonstration of such effects by
AM251, AM281, and AM630 on ionic currents. There is not
an effect on baseline currents in HEK293 cells, probably be-
cause not all the receptors are in a constitutively active state.
There is evidence that inactive hCB1 cannabinoid receptors
can form a stable complex precoupled with Gi/o, which
means that cannabinoid receptors would exist predominantly
in either a G-protein-precoupled inactive R–GGTP state or an
active R*–GGTP state (i.e., R, inactivated receptor; R*, acti-
vated receptor) (Vásquez and Lewis 1999); thus, it is neces-
sary to apply the agonist or the inverse agonist to observe
the effect on ionic currents.
In summary, our results indicate that endogenous K+ and
Ca2+ channels in HEK293 cells can be modulated by canna-
binoids. Some of the expressed hCB1 cannabinoid receptors
are in a constitutively active state in these cells, as previ-
ously described in other preparations. This gives us an addi-
tional tool for the study of the modulation of ionic currents
by cannabinoids.
Acknowledgements
This work was supported in part by a grant from
CONACyT-Mexico (I32814-N) and from Universidad de
Colima to C.V.
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References
Berjukow, S., Döring, F., Froschmayr, M., Grabner, M.,
Glossmann, H., and Hering, S. 1996. Endogenous calcium chan-
nels in human embryonic kidney (HEK293) cells. Br. J.
Pharmacol. 118: 758–754.
Bouaboula, M., Perrachon, S., Milligan, L., Canat. X., Rinaldi-
Carmona, M., Portier, M., Barth, F., Calandra, B., Pecceu, F.,
Lupker, J., Maffrand, J.P., Le Fur, G., and Casellas, P. 1997. A
selective inverse agonist for central cannabinoid receptor inhib-
its mitogen-activated protein kinase activation stimulated by in-
sulin or insulin-like growth factor 1. Evidence for a new model
of receptor/ligand interactions. J. Biol. Chem. 272: 22 330 –
22 339.
Chemin, J., Montiel, A., Perez-Reyes, E., Nargeot, J., and Lory, P.
2001. Direct inhibition of T-type calcium channels by the en-
dogenous cannabinoid anandamide. EMBO J. 20: 7033–7040.
Gebremedhin, D., Lange, A.R., Campbell, W.B., Hillard, C.J., and
Harder, D.R. 1999. Cannabinoid CB1 receptor of cat cerebral
arterial muscle functions to inhibit L-type Ca2+ channel current.
Am. J. Physiol. 276: H2085–H2093.
Gifford, A.N., and Ashby, C.R. 1996. Electrically evoked acetyl-
choline release from hippocampal slices is inhibited by the
cannabinoid receptor agonist, WIN 55212-2, and is potentiated
by the cannabinoid antagonist, SR 141716A. J. Pharmacol. Exp.
Ther. 277: 1431–1436.
Gifford, A.N., Tang, Y., Gatley, S.J., Volkow, N.D., Lan, R., and
Makriyannis, A. 1997. Effect of the cannabinoid receptor
SPECT agent, AM 281, on hippocampal acetylcholine release
from rat brain slices. Neurosci. Lett. 238: 84–86.
Hamill, O.P., Marty, A., Neher, E., Sakmann, B, and Sigworth, F.J.
1981. Improved patch-clamp techniques for high-resolution cur-
rent recording from cells and cell-free membrane patches.
Pflügers Arch. 391: 85–100.
Hoffman, A.F., and Lupica, C.R. 2000. Mechanisms of canna-
binoid inhibition of GABAA synaptic transmission in the hippo-
campus. J. Neurosci. 20: 2470–2479.
Kathmann, M., Bauer, U., Schlicker, E., and Gothert, M. 1999.
Cannabinoid CB1 receptor-mediated inhibition of NMDA- and
kainate-stimulated noradrenaline and dopamine release in the
brain. Naunyn-Schmiedeberg’s Arch. Pharmacol. 359: 466–470.
Katona, I., Sperlágh, B., Sík, A., Kafalvi, A., Vizi, E.S., Mackie,
K., and Freund, T.F. 1999. Presynaptically located CB1 canna-
binoid receptors regulate GABA release from axon terminals of
specific hippocampal interneurons. J. Neurosci. 19: 4544–4558.
Kim, D.J., and Thayer, S.A. 2000. Activation of CB1 cannabinoid
receptors inhibits neurotransmitter release from identified syn-
aptic sites in rat hippocampal cultures. Brain Res. 852: 398–405.
Landsman, R.S., Burkey, T.H., Consroe, P., Roeske, W.R., and
Yamamura, H.I. 1997. SR 141716A is an inverse agonist at the
human cannabinoid CB1 receptor. Eur. J. Pharmacol. 334: R1–
R2.
Mackie, K., Devane, W.A., and Hille, B. 1993. Anandamide, an en-
dogenous cannabinoid, inhibits calcium currents as a partial ag-
onist in N18 neuroblastoma cells. Mol. Pharmacol. 44: 498–503.
Mackie, K., Lai, Y., Westenbroek, R., and Mitchell, R. 1995.
Cannabinoids activate an inwardly rectifying potassium conduc-
tance and inhibit Q-type calcium currents in AtT20 cells
transfected with rat brain cannabinoid receptor. J. Neurosci. 15:
6552–6561.
MacLennan, S.J., Reynen, P.H., Kwan, J., and Bonhaus, D.W.
1998. Evidence for inverse agonism of SR 141716A at human
recombinant cannabinoid CB1 and CB2 receptors. Br. J.
Pharmacol. 124: 619–622.
© 2003 NRC Canada
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442
McAllister, S.D., Griffin, G., Satin, L.S., and Abood M.E. 1999.
Cannabinoid receptors can activate and inhibit G protein-
coupled inwardly rectifying potassium channels in a Xenopus
oocyte expression system. J. Pharmacol. Exp. Ther. 291: 618–
626.
Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C., and
Bonner, T.I. 1990. Structure of a cannabinoid receptor and func-
tional expression of the cloned cDNA. Nature (London), 346:
561–564.
Mundell, S.J., and Benovic, J.L. 2000. Selective regulation of en-
dogenous G protein-coupled receptors by arrestins in
HEK293cells. J. Biol. Chem. 275: 12 900 – 12 908.
Nakazi, M., Bauer, U., Nickel, T., Kathmann, M., and Schlicker, E.
2000. Inhibition of serotonin release in the mouse brain via
presynaptic cannabinoid CB1 receptors. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 361: 19–24.
Pan, X., Ikeda, S.R., and Lewis, D.L. 1996. Rat brain cannabinoid
receptor modulates N-type Ca2+ channels in a neuronal expres-
sion system. Mol. Pharmacol. 49: 707–714.
Pan, X., Ikeda, S.R., and Lewis, D.L. 1998. SR 141716A acts as an
inverse agonist to increase neuronal voltage-dependent Ca2+ cur-
rents by reversal of tonic CB1 cannabinoic receptor activity.
Mol. Pharmacol. 54: 1064–1072.
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Can. J. Physiol. Pharmacol. Vol. 81, 2003
Pertwee, R., Griffin, G., and Fernando, S. 1995. AM630, a compet-
itive cannabinoid receptor antagonist. Life Sci. 56: 1949–1955.
Ross, R.A., Brockie, H.C., Stevenson, L.A., Murphy, V.L.,
Templeton, F., Makriyannis, A., and Pertwee, R.G. 1999.
Agonist-inverse agonist characterization at CB1 and CB2 canna-
binoid receptors of L759633, L759656, and AM630. Br. J.
Pharmacol. 126: 665–672.
Schweitzer, P. 2000. Cannabinoids decrease the K+ M-current in
hippocampal CA1 neurons. J. Neurosci. 20: 51–58.
Twitchell, W., Brown, S., and Mackie, K. 1997. Cannabinoids in-
hibit N- and P/Q-type calcium channels in cultured rat
hippocampal neurons. J. Neurophysiol. 78: 43–50.
Van den Bossche, I., and Vanheel, B. 2000. Influence of
cannabinoids on the delayed rectifier in freshly dissociated
smooth muscle cells of the rat aorta. Br. J. Pharmacol. 131: 85–93
Vásquez, C., and Lewis, D.L. 1999. The CB1 cannabinoid receptor
can sequester G-proteins, making them unavailable to couple to
other receptors. J. Neurosci. 19: 9271–9280.
Yu, S.P., and Kerchner, G.A. 1998. Endogenous voltage-gated po-
tassium channels in human embryonic kidney (HEK293) cells.
J. Neurosci. Res. 52: 612–617.
© 2003 NRC Canada