Gris et al. Molecular Pain 2010, 6:33
A novel alternatively spliced isoform of the
mu-opioid receptor: functional antagonism
© 2010 Gris et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons At-
tribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Pavel Gris1, Josee Gauthier1, Philip Cheng1, Dustin G Gibson1, Denis Gris2, Oskar Laur3, John Pierson1,
Sean Wentworth1, Andrea G Nackley1, William Maixner1 and Luda Diatchenko*1
Background: Opioids are the most widely used analgesics for the treatment of clinical pain. They produce their
therapeutic effects by binding to μ-opioid receptors (MORs), which are 7 transmembrane domain (7TM) G-protein-
coupled receptors (GPCRs), and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised
by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH). In contrast to opioid
analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain
these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood.
Results: We recently discovered a new human alternatively spliced isoform of MOR (MOR1K) that is missing the N-
terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of
cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological
and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects.
In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide
(NO) release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Gαi/o
complex, MOR1K couples to the stimulatory Gαs complex.
Conclusion: The major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to
morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal
and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a
novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with
diminished ability to produce tolerance, OIH, and other unwanted side-effects.
The μ-opioid receptor (MOR) is the primary target for
both endogenous and exogenous opioid analgesics, medi-
ating basal nociception as well as agonist responses [1-4].
While opioids are the most frequently used and effective
analgesics for the treatment of moderate to severe clinical
pain, their prolonged use leads to a number of adverse
side-effects, including tolerance, dependence, and post-
dosing induced hyperalgesia, which is commonly referred
to as "opioid-induced hyperalgesia" (OIH) [5-7]. Several
hypotheses have been advanced to explain the mecha-
nisms underlying tolerance and OIH, including opioid
receptor downregulation, receptor desensitization, and/
or a decreased efficiency in G protein coupling. The cur-
rently held hypotheses fail to fully explain the mecha-
nisms that contribute to tolerance and OIH. For example,
receptor downregulation does not parallel the develop-
ment of tolerance to opioids . Additionally, the desen-
sitization of opioid receptor signaling following repeated
or prolonged opioid treatment  is unlikely to account
for opioid-induced tolerance as it has been reported to
suppress the development of tolerance . Thus, the
molecular mechanisms underlying opioid tolerance and
OIH require further investigation. One important, yet
underemphasized, cellular consequence of chronic opioid
treatment is the unmasking of excitatory signaling and
the suppression of the canonical inhibitory signaling
* Correspondence: Luda@UNC.edu
1 Center for Neurosensory Disorders, University of North Carolina at Chapel Hill,
Chapel Hill, NC, 27599, USA
Full list of author information is available at the end of the article
Gris et al. Molecular Pain 2010, 6:33
Page 2 of 10
The canonical signaling pathway for MOR agonists is
facilitated through a pertussis toxin (PTX)-sensitive
inhibitory G protein (Gαi/o), where analgesia reflects the
inhibition of synaptic transmission via inhibition of pre-
synaptic and postsynaptic voltage-gated Ca2+ channels
(VGCC) and/or a decrease in neuronal excitability via
activation of inwardly rectifying K+ channels. While opi-
oid-induced regulation of K+ current in sensory neurons
 and inhibition of adenyl cyclase (AC) have been
implicated in suppressing the activity of pronocicepitve
sensory primary neurons [15,16], the VGCC appears to
be the primary target underlying rapid opioid mediated
effects in these neurons [17,18]. This rapid inhibition of
VGCC reflects both a voltage-dependent and -indepen-
dent inhibition of high threshold channels[19-22]. MOR-
mediated inhibition of VGCC on central presynaptic ter-
minals of primary afferent nociceptors is thought to be
one of the primary mechanisms mediating analgesia at
the spinal level. However, opioid-induced hyperalgesic
responses have also been shown in animals and man fol-
lowing both acute and chronic dosing [23-26]. These
hyperalgesic effects are associated with concentration-
and time-dependent cellular excitation [15,16,27] as well
as with biphasic effects on cAMP formation and Sub-
stance P release [13,16,27-30]. Available evidence sug-
gests these excitatory effects reflect the activation of a
stimulatory G protein (Gαs) [11,31].
Using new bioinformatic approaches, we have recently
established the existence of previously undetected exons
within the human μ-opioid receptor gene OPRM1 .
These exons were discovered in a human genetic associa-
tion study that identified several single nucleotide poly-
morphisms (SNPs) associated with the individual
variability in pain sensitivity and responses to the MOR
agonist morphine. We found that exons carrying these
functional SNPs are spliced into a OPRM1 variant named
MOR1K that encodes for a 6TM rather than a canonical
7TM G-protein coupled receptor. The extracellular N-
terminus and first cytoplasmic domain are missing from
this isoform. Instead, MOR1K possesses a cytoplasmic N-
terminus followed by 6 transmembrane domains and C-
terminus homologous to MOR1. Thus, MOR1K should
retain the ligand binding pocket that is distributed across
the conserved TMH2, TMH3, and TMH7 domains 
and be capable of binding MOR agonists. Genetic analy-
ses revealed that allelic variants coding for higher
MOR1K expression are associated with greater sensitivity
to noxious stimuli and blunted responses to mor-
phine. This relationship is opposite to that expected
for MOR and suggests a pronociceptive function for
MOR1K. We thus hypothesized that MOR1K contributes
to hyperalgesic effects of MOR agonists through the acti-
vation of cellular excitatory pathways. To test this
hypothesis, we first characterized tissue-specific expres-
sion levels of MOR1K, its cellular localization, and ago-
nist binding capacity to confirm potential functionality of
this new receptor isoform. We then employed pharmaco-
logic and molecular biologic methods to measure and
compare intracellular cAMP and Ca2+ levels as well as
nitric oxide (NO) release in response to stimulation of
canonical MOR1 and alternative MOR1K isofoms.
Immunoprecipitation experiments were carried out to
test if the MOR1K isoform couples to the stimulatory Gαs
Results And Discussion
MOR1K expression and binding pattern
First, we characterized the relative expression of MOR1K
in human brain and spinal cord as well as peripheral leu-
kocytes using real-time PCR (RT-PCR). Results from RT-
PCR revealed that MOR1K is expressed in the frontal
lobe, medulla oblongata, insula, nucleus accumbens,
pons, spinal cord, and dorsal root ganglion (DRG)
(Fig.1A-B). These brain regions are known to also express
MOR1 and contribute to the pharmacological effects of
MOR agonists. MOR1K expression was not observed in
the connective tissue surrounding DRG or in peripheral
leukocytes. Additionally, MOR1K was expressed at high
levels in the human neuroblastoma cell lines Be2C and
SH-SY5Y, while no expression was observed in monkey
kidney COS-1 and human astrocytoma H4 cell lines.
These data suggest that human MOR1K expression is
restricted to neuronal cells. As the highest relative
expression levels are in transformed neuronal cell lines,
MOR1K expression is likely to be suppressed in native
To characterize the cellular location and function of the
new human MOR1K isoform, expression vectors with
cloned coding regions of MOR1 (7TM) or MOR1K
(6TM) receptor isoforms, with or without MYC (MOR1)
or FLAG (MOR1K) tags at their N-termini, were tran-
siently transfected into a cell line that expresses endoge-
nous MORs (i.e., Be2C human neuroblastoma cell line)
and a cell line that does not express endogenous MORs
(i.e., african green monkey kidney COS1 and human
embryonic kidney (HEK293) cells). Overexpression of
MOR1K in mammalian cells revealed that this 6TM
receptor is not expressed at the cell membrane, but
instead is retained in the intracellular compartment
(Fig.1C). As intracellular localization of several receptor
systems does not prevent receptor-mediated signalling
[34,35], we examined whether the intracellulary localized
MOR1K binds MOR ligands using flow cytometry  to
measure binding of fluorescently labeled naloxone
(Fig.1D-E). Be2C cells transfected with either MOR1 or
MOR1K retained a significantly higher proportion of flu-
orescently-labeled naloxone in comparison with cells
Gris et al. Molecular Pain 2010, 6:33
Page 3 of 10
transfected with an empty vector control. This retention
was abolished in the presence of excess of unlabelled
naloxone demonstrating the specificity of binding. These
data suggest that MOR ligands, such as naloxone, can
cross the plasma membrane and MOR1K is a functional
intracellular receptor that binds MOR ligands. Although
this set of studies has employed naloxone, a movement of
morphine across the cellular membrane by means of
active transport or passive diffusion has also been shown
[37,38]. Furthermore, the rate of passive diffusion of weak
base ligands like morphine across the cell membrane can
be examined by the Henderson-Hasselbalch equation
[39,40], estimating that approximately 25% of the total
amount of morphine present in the medium can diffuse
across membranes to enter or exit cells.
Effect of MOR1K activation on cAMP, Ca2+ and NO levels
Next, we characterized the cellular effects of MOR1K
stimulation using cAMP, Ca2+, and NO signaling assays.
It is known that stimulation of the MOR1 leads to the dis-
sociation of the heterotrimeric Gαi/o -protein complex,
where release of the α subunit results in the inhibition of
the adenylyl cyclase/cAMP pathway and release of the βγ
subunits inhibits VGCC [15,16]. Therefore, the cellular
characterization of cAMP accumulation and intracellular
Ca2+ levels were used to assess the functional effects of
MOR1K activation [16,27,41-44]. In agreement with an
inhibitory function of MOR1, COS1 cells transfected
with this isoform demonstrated a significant decrease in
forskolin-induced cAMP levels following treatment with
1 μM of morphine. In contrast, COS1 cells transfected
with MOR1K did not show a morphine-dependent
decrease in forskolin-induced cAMP levels. Instead, they
showed a trend towards an increase in cAMP levels
(Fig.2A). Furthermore, COS1 cells transfected with
MOR1K showed a substantial increase in intracellular
Ca2+ release following morphine treatment, which was
Figure 1 MOR1K expression and binding pattern. (A) The schematic diagram illustrates the exonic composition and relative positions of PCR prim-
ers designed to amplify the major MOR1 isoform and the newly identified alternative MOR1K isoform. The relative positions of translation initiation start
and stop codons are designated by ATG and TGA, respectively. The predicted protein structure of MOR1 and MOR1K isoforms is schematically present-
ed. Translation of the MOR1K variant results in a 6TM receptor, truncated at the N-terminus. (B) Real-time PCR was performed on total RNA samples
from the human brain regions known to express MOR1. Primers specific for exons 1 and 2 were used to measure MOR1 and primers specific for exons
13 and exon 2 were used to measure MOR1K. GAP3DH was used as a control for cDNA loading and PCR efficiency. (C) Confocal images of C-ter-
minally MYC-tagged MOR1 or FLAG-tagged MOR1K overexpressed in HEK293 cells and stained with either Anti-MYC-Tag Antibody (Alexa Fluor 647
Conjugate) or Anti-DYKDDDDK Tag Antibody (Alexa Fluor 555 conjugate). Cells transfected with MOR1 showed membrane expression of receptor,
while cell transfected with MOR1K express receptor only intracellular. (D) Confocal images of C-terminally FLAG-tagged MOR1K overexpressed in Be2C
cells and stained with either Anti-FLAG M2 Antibody Alexa Fluor Conjugate or fluorescent-labeled naloxone (FNAL). Cells transfected with MOR1K
showed intracellular retention of FNAL that co-localized with antibody-labeled receptor. (E) The binding of naloxone to MOR1K was assessed using
flow cytometry to measure FNAL retention. Be2C cells transfected with either MOR1 or MOR1K isoforms showed increased retention of FNAL at con-
centrations of 0.1 and 1 μM. FNAL retention was abolished in the presence of 10 μM unlabelled naloxone (Nal). In panel E, data are presented as mean
+ SEM. *P < 0.05 different from controls).
Fluorescence Intensity (RFU)
M OR1 exons 1-2
M OR1K exons 13-2
MOR1 exons 1-2
MOR1K exons 13-2
Gris et al. Molecular Pain 2010, 6:33
Page 4 of 10
not observed in MOR1 expressing cells (Fig. 2B). These
findings were replicated in Be2C neuroblastoma cells
Because VGCC appears to be the primary target under-
lying the rapid inhibitory effects of opioids[17,18] and
morphine stimulation of MOR1K increases intracellular
Ca2+ levels in COS1 cells (Fig.2B), we examined the dose-
dependent regulation of Ca2+ levels in neuroblastoma
BE2C cells transiently transfected with MOR1K (Fig.2C).
In BE2C cells, stimulation of MOR1K with morphine
produced a robust dose-dependent increase in Ca2+ levels
that was blocked by naloxone. A moderate increase in
morphine-evoked Ca2+ levels was also observed in Be2C
cells transfected with MOR1 or empty vector, however
this was likely due to the high endogenous expression of
MOR1K in this cell line (Fig.1B). Consistent with this
view, the increase in Ca2+ levels in cells transfected with
MOR1K was significantly higher than the increases
observed in cells transfected with either MOR1 or empty
vector, all morphine-dependent Ca2+ increases were sen-
sitive to opioid-receptor blockage with naloxone and
MOR1-dependent morphine-evoked increases in Ca2+
were not observed in COS1 cells that do not express
endogenous MOR1K (Fig. 1B, 2B).
Because the main cellular response mediated by
another reported 6TM isoform, MOR-3, is a morphine-
dependent increase in nitric oxide (NO) production ,
we examined the effects of morphine on NO release from
immortalized cell lines transfected with MOR1 or
MOR1K isoforms (Fig.3). There were marked differences
between MOR1 and MOR1K with respect to morphine-
induced NO production in transfected Be2C cells. In
contrast to MOR1, activation of MOR1K with morphine
produced a substantial dose-dependent increase in NO
that was blocked by naloxone (Fig. 3A). Also, the time
required for NO to reach its maximum level occurred
more rapidly in MOR1K relative to MOR1 transfected
cells. For MOR1K expressing cells, maximum NO pro-
duction (16 pM) occurred at ~25 seconds and returned to
a baseline at ~80 sec following 1 μM morphine adminis-
tration. For MOR1 expressing cells, maximum NO pro-
duction (7.5 pM) occurred at ~50 sec after administration
of 1 μM morphine (Fig. 3C). Similar results were obtained
in COS1 cells (data not shown). Importantly, morphine-
dependent release of NO promotes a reduction in opioid
analgesia as well as an increase in analgesic tolerance and
OIH in animal models [46-49].
MOR1K coupling to G-protein complexes
GPCR signaling is initiated by activating heterotrimeric
G-protein complexes. Uncoupling of Gαi-Gαo inhibits AC
resulting in decreased cAMP production, while the
release of βγ subunits inhibits Ca2+ channels so as to
Figure 2 MOR1K activation stimulates cAMP and Ca2+. COS1 (A,B) or BE2C (C) cells were transiently transfected with MOR1K, MOR1, or empty vec-
tor control expressing constructs. (A) Forskolin (FSK, 10 μM) was used to increase cAMP levels prior to morphine treatment. Following morphine treat-
ment, cells expressing MOR1 exhibited reduced cAMP levels, while those expressing MOR1K did not. In fact, cells expressing MOR1K exhibited a trend
towards an increase in cAMP levels. (B) Following morphine treatment, COS1 cells expressing MOR1K exhibited substantial increases in Ca2+ levels,
while those expressing MOR1 did not. (C) Stimulation of MOR1K with morphine produced a robust dose-dependent increase in Ca2+ levels in Be2C
cells. This increase was significantly different from the moderate increase in morphine-evoked Ca2+ levels observed in Be2C cells transfected with
MOR1 or empty vector, which were likely due to the high endogenous expression of MOR1K in Be2C cells. For all panels, both MOR1 and MOR1K,
morphine-dependent effects were antagonized in the presence of naloxone (0.1 μM). Data are presented as mean ± S.E.M from at least 6 experiments.
$P < 0.05 different from control and *P < 0.05 different from control and MOR1.
morphine + - + + + + + +
FSK - + + + + + + -
NAL - - - - + - + -
morphine - + - + - +
Ca++ Increase (%)
Ca++ Increase (%)
-8-7 -6 -5
Gris et al. Molecular Pain 2010, 6:33
Page 5 of 10
inhibit neural activity. Conversely, uncoupling of Gαs and
Gαq subunits results in increased cAMP production and
cellular excitation. Electrophysiological studies of the
effects of opioids on isolated nociceptive-like dorsal root
ganglion (DRG) neurons provides in vivo evidence that
the inhibitory effects (e.g. shortening of the Ca2+-depen-
dent component of the action potential duration and
inhibition of transmitter release) are mediated by Gαi/o-
dependent pathways. In contrast, the excitatory effects
(e.g. prolongation of the action potential duration and
stimulation of transmitter release) are mediated by Gαs -
dependent pathways[11,31]. Since activation of MOR1K
results in the intracellular accumulation of Ca2+ and
showed a tendency to increase cAMP levels, we exam-
ined whether MOR1K couples to Gαs, versus Gαi. A set of
co-immunoprecipitation experiments was conducted to
elucidate MOR1K's coupling partner (Fig. 4). As
expected, MOR1 co-immuprecipitated with Gαi and Gαo.
In contrast, MOR1K co-immuprecipitated with Gαs.
These results are consistent with the observation that
treatment with pertussis toxin does not block MOR1K-
dependent increases in intracellular Ca2+ (Fig. 4). Cou-
pling with Gαq was not observed for either MOR1 or
Molecular and medical implications
Our results have very broad basic molecular and medical
implications. First, they significantly contribute to our
emerging understanding of the molecular and cellular
biology of MOR receptor and GPCRs in general. MOR1K
is one of several human and mouse MOR alternatively
spliced variants coding for a truncated 6TM receptor
lacking an extracellular N-terminal domain and trans-
membrane domain I [45,50,51]. To date, the functional
significance of truncated 6TM MOR receptors has not
been examined. The results of our studies are the first to
show that activation of this 6TM MOR isoform results in
increased production of mediators (Ca2+ and NO) associ-
ated with cellular excitation. Furthermore, immunopre-
cipitation experiments revealed that the MOR1K 6TM
receptor couples to Gαs rather than Gαi. Unlike the stim-
ulation of the MOR1 7TM, which inhibited the produc-
tion of cAMP, morphine stimulation of the MOR1K 6TM
failed to inhibit cAMP production and instead tended to
increase cAMP production. The finding that MOR1K
receptor couples with Gαs suggests that we were not able
to fully assess the extent of the cAMP regulation by
MOR1K, which will require additional optimization of
the cellular conditions. However, the robust effect of
Figure 3 MOR1K activation stimulates NO production. Be2C cells were transiently transfected with MOR1K or MOR1 expressing constructs. (A)
MOR1K produced robust concentration-dependent increases in the release of NO. (B) The time course associated with morphine-induced (1 μM) NO
release was markedly different in response to stimulating MOR1K and MOR1. NO production was blocked by pretreatment with naloxone (0.1 μM)
and was significantly different from the respective controls. Data are presented as mean + SEM. *P < 0.05 different from controls.
NO production (pM)B
-10-9 -8 -7-6-5
NO production (pM)
Gris et al. Molecular Pain 2010, 6:33
Page 6 of 10
morphine on MOR1K-dependent Ca2+ and NO release, in
conjunction with a primary role of VGCC in mediating
the analgesic effects of opioids [17,18] and the observa-
tion that MOR1K couples to Gαs, supports the view that
MOR1K, and perhaps other 6TM isoforms, function to
counteract the cellular actions mediated by canonical
Although there are several examples in other receptor
systems where truncated isoforms modulate the activity
of [52-56] or display opposite biological activity relative
to the canonical receptor , the functional significance
of this interaction has not been fully appreciated. Because
GPCRs are the major targets for therapeutic drugs com-
monly used in clinical practice, and only a few other 6TM
GPCRs isoforms have been reported (e.g., histamine
H3, prostanoid  and adrenergic alpha1A 
receptors), our data open up a new vista for a fuller inves-
tigation of the functional importance of 6TM truncated
GPCR isoforms in GPCR signaling and drug responses.
The observed intracellular localization of the MOR1K
receptor isoform, while relatively rare, is well described
for other functional receptor systems. One well known
example is the sigma receptor (Sigma-1) that reside pri-
marily at the endoplasmic reticulum and whose ligands
include cocaine, (+)-benzomorphans like (+)-pentazo-
cine, (+)N-allyl-normetazocine, and endogenous neuros-
teroids like progesterone and pregnenolone sulfate .
Many pharmacological and physiological actions have
been attributed to sigma-1 receptors. These include the
regulation of IP3 receptors and calcium signaling at the
endoplasmic reticulum, mobilization of cytoskeletal
adaptor proteins, modulation of nerve growth factor-
induced neurite sprouting, modulation of neurotransmit-
ter release and neuronal firing, modulation of potassium
channels as a regulatory subunit, promotion of psycho-
stimulant-induced gene expression, and blockade of
spreading depression . Among GPCRs, a well known
example of an intracellularly located receptor is GPR30
that is localized to the endoplasmic reticulum, where it
specifically binds estrogen. Activation by estrogen results
in intracellular calcium mobilization and synthesis of
phosphatidylinositol 3,4,5-trisphosphate (IP3) in the
nucleus . However, the possibility that MOR1K cell
surface expression is very transient or/and requires a
chaperone for plasma membrane co-expression [60,61]
can not be ruled out.
Finally, our results also suggest that MOR1K may medi-
ate the molecular processes that underlie OIH and possi-
bly pharmacological tolerance commonly observed in
response to opioids. Because stimulation of MOR1K
results in the production of excitatory mediators such as
Ca2+, and NO that have been shown to contribute to tol-
erance and OIH [16,46-49,62], the up-regulation of this
isoform or desensitization of the major 7TM MOR1 iso-
form in response to prolonged opioid administration may
result in a change in the balance between 7TM MOR- and
6TM MOR1K-mediated activities and thus dictate the
effects of MOR stimulation on physiological processing
of nociceptive information. Thus, further investigation of
MORK1 expression and function, in conjunction with
major MOR1 isoform expression and function under
chronic opioid exposure in animal and human models is
Figure 4 MOR1K couples to Gαs. COS1 cells were transiently transfected with MYC-tagged MOR1 or FLAG-tagged MOR1K constructs. (A) Iimmuno-
precipitation was performed with anti-FLAG (raised in rabbit) and anti-MYC (raised in rabbit) antibodies. Immunoblotting was conducted with specific
anti-Gα antibodies as described. The MOR1K-FLAG construct was found to couple to Gαs, while the MOR1-MYC construct coupled only to Gαi/o. The
exposure time for MOR1K-FLAG experiments were ~2 times longer to see band intensities comparable to MOR1-MYC. (B) Consistent with Gαs binding
to the MOR1K isoform, morphine-mediated increases in Ca2+ levels were not prevented by pre-treatment of MOR1K expressing cells with PTX (100
ng/ml for 15min).
Ca Increase (%)
Gris et al. Molecular Pain 2010, 6:33
Page 7 of 10
In summary, MOR agonists are among the most widely
prescribed analgesics for both acute postoperative pain
and chronic pain conditions; yet, there are substantial
drug-induced side-effects of which we have very limited
understanding. Our results provide substantial evidence
that the 6TM MOR1K isoform is not just another alterna-
tively-spliced form of MOR1, but instead it represents a
functional receptor that contributes to the net cellular
response of MOR agonists by facilitating excitatory
effects. Thus, MOR1K may represent a new molecular
target that mediates OIH, and analgesic tolerance. Eluci-
dating the biological and cellular properties of 6TM and
7TM MOR receptor variants may ultimately lead to the
identification and development of new classes of opioid
analgesics, such as 7TM-selective agonists and/or 6TM-
slelective antagonists, that show a high degree of analge-
sic efficacy with fewer treatment limiting side-effects.
MOR1K and MOR1 expression constructs were sub-
cloned into pIRES2-EGFP (Clontech) expression vector
using SacI/SacII restriction sites. MOR PCR products
were generated using the following primers: for MOR1
CCCCACGAAC and ATCCCCGCGGTTAGGGCA
CTTC and AAGCTTGCCACCATGAAGACTGCCAC-
CAACATCTACATTTTC). The following primers were
used to fuse Myc and Flag sequences to 5'end of MOR1
and MOR1K: ATATCGAGCTCGCCACCATGG
ACAG and ATATCGAGCTCGCCACCATGGAT
tively. All constructs were sequence verified.
Be2C, COS1, SH-SY5Y, HEK293 and H4 cell lines were
obtained from ATCC. The cells were grown to 90% con-
fluence and transfected with MOR1K, MOR1 or GFP
expression constructs with Lipofectamine 2000 reagent
(Invitrogen) according to manufacturer's guidelines.
RNA from cell lines was extracted using a Qiagen RNeasy
kit. The human tissues RNA samples were purchased
from Takara Bio. The 2-3 μg of RNA was treated with
TURBO-DNA free (Ambion) and reverse transcribed
using SuperScript III Reverse Transcriptase (Invitrogen)
kits. The cDNA was amplified with SYBR Green PCR
Master mix (Applied Biosystems Inc) using forward and
reverse PCR primers using 50°C for 2 min and 95°C for 10
min followed by 40 cycles of 95°C for 15 sec and 60°C for
1 min. 7900HT Fast real-Time PCR system (ABI) was
used for measuring RNA transcripts amplification. The
same exon 2-specific reverse primer (GCCAGAG-
CAAGGTTGAAAATG) was used in combination with
either exon 1-specific forward primer (CTTCCTGGT-
CATGTATGTGATTGTC) or exon 13 -specific forward
HEK293 or Be2C cells were fixed with paraformaldehyde
(4%) and subsequently permeabilized with Triton X-100
(.003%). Permeabilized cells were stained using Anti-
Myc-Tag Antibody (Alexa Fluor 647 Conjugate), Anti-
DYDDDDK Tag Antibody (Alexa Fluor 555 Conjugate)
(Cell Signaling), Anti-FLAG M2 Antibody Alexa Fluor
Conjugate or fluorescent-labeled naloxone (FNAL,
Sigma) and coated with Prolong Gold Anti-Fade Reagent
(Invitrogen). Images were obtained on an Olympus
FV500 confocal laser scanning microscope with excita-
tion wavelengths of 546 nm and 647 nm.
Be2C cells were stained on ice in PBS containing 2% FBS.
Fluorescence intensities were acquired using a CyAn
ADP high resolution cytometer (DAKO, Fort Collins,
CO). Concentration of unlabeled naloxone was main-
tained at 10 μM throughout the experiment. Dead cells
were excluded using forward and side scatter characteris-
tics and at least 2 × 104 live cells were acquired. The
intensity of staining was expressed in arbitrary units of
fluorescence. Four independent experiments were per-
formed in triplicate. Mean values were compared using
two way ANOVA followed by Tukey's test.
For the cAMP accumulation assay, MOR1 or MOR1K
expressants were plated on 12 well plates and grown to
90% confluency. On the day of sample preparation, cells
were washed with DMEM to remove serum and incu-
bated with serum-free DMEM containing the phosphodi-
esterase inhibitor IBMX (100 μM)(Sigma) for 30 min.
Morphine was then added and cells incubated for a fur-
ther 15 min. Following this, forskolin FSK (50 μM) was
added to the wells and the cells were incubated for 15 min
to stimulate cAMP production. DMSO alone was used as
a vehicle control. After incubation, reactions were termi-
nated by aspiration of the medium and addition of 0.1 M
HCl followed by 20 min incubation at room temperature.
After centrifugation of the cell samples at 10,000 × g for
10 min, protein content of the supernatant was assessed
and the samples were diluted to protein concentrations of
Gris et al. Molecular Pain 2010, 6:33
Page 8 of 10
20 μg/ml. The levels of cAMP were determined using an
enzyme immunoassay (EIA) cAMP EIA kit (Cayman
Chemical) according to the manufacturer specifications.
Be2C human neuroblastoma cells were grown to near
confluence in black 96-well poly-D-lysine coated plates.
The cell cultures were grown in DMEM/F12 media. The
indicator Fluo-4 NW dye (Invitrogen) was prepared as
outlined in manufacturer instructions. 100 μL of Fluo-4
NW dye was added to each well. The plate was then incu-
bated with the lid on at 37°C for 30 minutes, then at room
temperature for an additional 30 minutes. The fluores-
cence was measured using Victror-3 (Perkin Elmer)
microplate reader with settings for emission at 515 nm
and excitation at 500 nm.
Nitric oxide (NO) measurement
Cells were grown in DMEM/F12 supplemented with 10%
FBS at 37°C. NO release from the transfected and
untransfected cell lines was directly measured using an
NO-specific amperometric probe (Innovative Instru-
ments). The system was calibrated daily by nitrosothiol
donor S-nitroso-N-acetyl-D,L-penicillamine (World Pre-
cision). The amperometric probe was allowed to equili-
brate for at least 10 min before being transferred to the
well containing the cells. Morphine-stimulated NO
release was evaluated in response to increasing morphine
concentrations in the range of 10-5-10-9 M. Each experi-
ment was repeated four times along with a control (cells
transfected with vector alone).
The cells were lysed using RIPA buffer and centrifuged at
maximum RPM for 20 minutes. Supernatants were col-
lected and used for co-IP experiments. Following the
overnight incubation on the rotary shaker at 4°C with
anti-FLAG (MOR1K) or anti-MYC (MOR1) antibodies,
the beads (Pierce) were added, and the samples were
incubated for 6 hours at 4°C. The samples were then cen-
trifuged at 14000 rpm for 10 min, the supernatants were
discarded, and the beads were re-suspended in 50%
RIPA/PBS buffer. The procedure was repeated 3 times.
The samples were then boiled and run on 12% SDS-PAGE
gels (Invitrogen) and transferred to nitrocellulose mem-
branes (Hybond ECL; GE Healthcare Bio-Sciences). After
blocking overnight at 4°C with 5% non-fat dried milk in
Tris-buffered saline with Tween 20 (150 mM NaCl, 20
mM Tris-HCl, pH 7.5, 0.1% Tween 20), the membranes
were probed with primary antibodies [anti-Gαs (K-20):
sc-823, anti-Gαi (C-10): sc-262 and anti-Gβ (T-20): sc-
378; Santa Cruz Biotechnology, 1:1,000] overnight at 4°C.
After washing in TBS-T (three times, 5 min each), the
blots were incubated for 4 hours at room temperature
with horseradish peroxidase-conjugated secondary anti-
body (1:15,000; GE Healthcare Bio-Sciences). All anti-
bodies were diluted in blocking buffer. The antibody-
antigen complexes were detected using the ECL system
(Amersham) and visualized with photosensitive film
List of abbreviations
OIH: opioid-induced hyperalgesia; MOR: μ opioid recep-
tor; 7TM: 7-transmembrane;
brane;Ca2+: calcium; cAMP:
monophosphate; FSK: forskolin; IBMX: 3-isobutyl-1-
methylxanthine; EIA: Enzyme immune assay; PTX: Per-
tussis toxin; VGCC: voltage-gated calcium channel; K+
channel: potassium channel; AC: Adenyl cyclase; SNP:
single nucleotide polymorphism; OPRM1: μ opioid
receptor gene; TMH: trans-membrane domain; DRG:
dorsal root ganglion; NO: nitric oxide; GPCR: G-protein
coupled receptor; IRES: internal ribosome entry site;
EGFP: enhanced green fluorescence protein; RIPA buffer:
Radioimmunoprecipitation assay buffer; ECL: enhanced
Financial competing interests: Luda Diatchenko, William Maixner, Pavel Gris and
Josee Gauthier are listed as inventors on a pending patent relating to the con-
tent of the manuscript. During the last five years, Drs. Maixner and Diatchenko
have received consulting fees from a company they cofounded, Algynomics
Inc. This company has the rights to the pending patent in areas related to
OPRM1 and work described in this manuscript. Drs. Maixner, Diatchenko, and
Nackley are also equity stock holders in Algynomics. Algynomics has not pro-
vided funding for the work described in this manuscript. Non-financial compet-
ing interests: Other than current and recently submitted NIH grant applications
there are no other non-financial competing interests
PG participated in the design of the study, carried out the NO and Ca2+
response studies, the, co-IP and labeled naloxone confocal imaging studies,
and drafted the figures and manuscript. JG carried out expression studies, and
helped draft the figures and manuscript. PC carried out Ca2+ assays. DGG car-
ried out co-IP assays and Western blotting. DG designed and performed flow
cytometry experiments. OL designed the expression clones and performed
cloning and the sequence alignment. JP carried out cAMP assays. SW carried
out the confocal microscopy imaging. AGN participated in its design and coor-
dination and helped draft the manuscript. WM conceived of the study, partici-
pated in its design and helped to draft the manuscript. LD conceived the study,
participated in its design and coordination, and helped to draft the manu-
script. All authors read and approved the final manuscript.
This work was supported in part by NIH/NIDCR, NIH/NINDS and NIH/NCRR
grants RO1-DE16558 (LD, WM), UO1-DE017018 (WM, LD), NS41670 (WM), PO1
NS045685 (WM, LD), KL2RR025746 (AGN) and UL1RR025747 (AGN).
1Center for Neurosensory Disorders, University of North Carolina at Chapel Hill,
Chapel Hill, NC, 27599, USA, 2Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA and
3Yerkes Research Center - Division of Microbiology, Emory University, Atlanta,
GA, 30329, USA
Received: 19 March 2010 Accepted: 2 June 2010
Published: 2 June 2010
This article is available from: http://www.molecularpain.com/content/6/1/33 © 2010 Gris et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Pain 2010, 6:33
Gris et al. Molecular Pain 2010, 6:33
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Cite this article as: Gris et al., A novel alternatively spliced isoform of the mu-
opioid receptor: functional antagonism Molecular Pain 2010, 6:33