(Received for publication, September 8, 1994; and in revised form, October 27, 1994)
From the
In Xenopus oocytes expressing the rat mu receptor and
the G protein-gated, inwardly rectifying K channel
(known as KGA or GIRK1), application of
[DAla
,MePhe
,Glyol
]enkephalin),
a mu opioid agonist,evoked a dose-dependent increase in K
conductance. With sustained agonist exposure, the amplitude of
the response decayed with a t
of 8 ± 2
min. In oocytes coexpressing the mu and 5HT1A receptors with GIRK1,
stimulation of either receptor resulted in heterologous desensitization
of the subsequent response to the other. Injection of guanosine
5`-O-(thiotriphosphate) (1 mM) increased the basal
GIRK1 activity and the total response to the application of agonist,
but did not affect the rate of desensitization. Basal channel activity
in the absence of agonist also desensitized at the same rate when the
oocytes were exposed to high K
(96 mM)
solution. The above results indicate that the desensitization of the
response occurred at a site downstream of the receptor, possibly at the
channel. The rate of desensitization was not significantly altered by
any of the following treatments: removal of external
Ca
, preloading the oocytes with
1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic
acid-tetra-(acetoxymethyl)-ester (0.5-1 mM), elevation
of cAMP levels, treatment with phorbol esters (1 µM),
staurosporine (0.5 µM), okadaic acid (1 µM),
or cytochalasin B (0.5 µM). These results suggest that
desensitization may not involve a calcium or phosphorylation-dependent
mechanism.
Recently, a G protein-activated inwardly rectifying potassium
channel (KGA or GIRK1) was cloned from rat
atrium(1, 2) . Northern blot analysis indicated
abundant expression of the channel in the
brain(2, 3) , and a virtually identical clone was
isolated from a rat brain library(3) . Opioid receptors were
among the several cloned G protein-coupled receptors (including
muscarinic ACh, 5HT1A) shown to functionally couple to the GIRK1
channel coexpressed in Xenopus oocytes(3, 4) . The coupling of the G
protein-gated inwardly rectifying K channel to
neurotransmitter receptors in cardiac cells and in neurons occurs
through a membrane-delimited pathway that does not involve cytoplasmic
intermediates and is mediated by heterotrimeric G proteins (for review,
see (5, 6, 7) ). Several reports have
alternatively implicated the GTP-bound G
subunit (8, 9, 10) or the
complex as the
channel activating component(11, 12, 13) .
The activation of the opioid receptors has been shown to modulate
neuronal excitability and inhibit neuronal firing in various portions
of the nervous system (see (14) ). Stimulation of the mu opioid
receptor results in a membrane hyperpolarization induced by the
activation of an inwardly rectifying K conductance in
both locus coeruleus and hippocampal
neurons(15, 16, 17) . Several lines of
evidence suggest that the cloned GIRK1 channel is identical or very
similar to the K
channel linked to the opioid receptor
and other neurotransmitter receptors. The biophysical properties of the
GIRK1 clone, the inwardly rectifying potassium channels opened by
muscarinic agonists in the heart, and opioids in the locus coeruleus,
are indistinguishable as determined by unit conductance, duration of
the closed states, and inward rectification
properties(2, 3, 16, 18, 19) .
In addition, it has been shown in Xenopus oocytes that the
coupling of the GIRK1 clone is membrane delimited (2, 3) and is partially sensitive to pertussis
toxin(3, 4) . Expression of the cloned opioid
receptors (see (20) ) in Xenopus oocytes allows the
analysis of the signal transduction process.
Sustained exposure of
neurons to a neurotransmitter often leads to a desensitization of the
response, and a prominent feature of opioid drugs is their ability to
induce tolerance and dependence in humans and experimental animals. In
addition to changes at the level of the receptor, changes in associated
and effector macromolecules are involved in the desensitization
process(21, 22) . We report here that the inwardly
rectifying K conductance can be activated by DAMGO, (
)a mu agonist, in Xenopus oocytes expressing the
mu opioid receptor and GIRK1 channel. The evoked response decays, and
desensitization of the response to the agonist is likely to be a
consequence of channel inactivation.
Changing from ND96 to the hK
solution was accompanied by the development of an inward current
(I) (Fig. 1A). A small I
(<100 nA) was observed in native, uninjected oocytes. In
oocytes injected with the mu receptor cRNA alone and in native oocytes,
I
was not significantly different nor increased by mu
agonist. I
in oocytes injected with GIRK1 cRNA alone was
larger than in uninjected oocytes, and I
was even greater
in oocytes expressing both the channel and receptor proteins (data not
shown). Thus, in oocytes expressing GIRK1, I
was composed
of intrinsic potassium currents as well as basally activated GIRK1
currents. GIRK1-mediated currents can be distinguished by block using
external Ba
(300 µM)(1) .
Ba
did not significantly change the amplitude of
I
in native oocytes or in oocytes injected with receptor
cRNA alone, but in oocytes expressing the GIRK1 channel or both channel
and receptor, Ba
reduced all inward currents evoked
in hK to levels seen in the native oocytes (Fig. 1A).
Oocytes expressing both the mu opioid receptor and GIRK1 gave rise to
an increase in the inward current upon application of the mu receptor
agonist DAMGO (I
) (Fig. 1A).
The response evoked by DAMGO was dose dependent with an EC
of 3 nM (Fig. 1B) and was completely
blocked by the opioid receptor antagonist naloxone (Fig. 1A).
Figure 1:
Coupling of the mu
opioid receptor to the G protein-activated inward rectifier
K channel, GIRK1. Oocytes were injected with cRNAs for
the mu opioid receptor and the GIRK1 channel. A,
representative current trace showing the change in current following
solution changes are shown above. In this and all subsequent figures,
the current traces were recorded at a holding potential of -80
mV. DAMGO (1 µM) naloxone (10 µM), and
BaCl
(300 µM) were applied at the times shown. B, response to DAMGO (peak
I
) was measured as a function of
concentration in oocytes injected with 1 ng each of GIRK1 and mu
receptor cRNA. Each oocyte was exposed to only a single dose of DAMGO,
and each point represents the mean response (± S.E.) from three
or four oocytes.
Figure 2:
Reduction of the evoked current response
of the GIRK1 channel. Oocytes were injected with mu receptor and GIRK1
channel cRNAs. A, a large inward current response was obtained
by exposing a representative oocyte to 1 µM DAMGO in hK.
Membrane currents with voltage steps of 20 mV ranging from -160
mV to +40 mV were recorded at peak inward current and
approximately 20 min later when the inward current had desensitized
(shown as time points 1 and 2 on the trace). Voltage
steps were also performed after the application of 300 µM Ba. B, the current flowing through the
GIRK1 channel at any potential was calculated as the current obtained
after the subtraction of the inward current, at the same potential, in
the presence of 300 µM Ba
. The
current-voltage relationships obtained in this manner at the peak
response (1) and when the response had attenuated (2). C, application of DAMGO concentrations between
0.1 nM and 1 nM produces peak responses (I
) that were less than 350 nA while peak
responses greater than 1 µA were elicited with 1 µM DAMGO. The rate of desensitization was independent of the
amplitude of the peak response. D, to determine the rate of
densensitization of a population of oocytes, the evoked inward GIRK1
current (I) was normalized to the amplitude of the peak
response (I
) for each oocyte as a function
of time. The normalized decay curve from each oocyte in the population
was averaged to arrive at the mean decay curve for the population which
was then fitted by a single order exponential. E, a
representative current trace evoked in an oocyte expressing ROMK1 (the
results shown were replicated three times).
The inward current through the
Ba-insensitive endogenous conductance of the oocyte
did not significantly attenuate (data not shown). Extrapolating to t = infinity for the decay exponential, the activated
current reduced to the endogenous Ba
-insensitive
component. These results suggest that the decay of the inward current
was caused by a reduction of current carried by the GIRK1 channels
alone. Subsequent analyses of the response desensitization were made
after subtraction of the Ba
-insensitive component
determined at the end of the measure (Fig. 2A).
The
current-voltage relationship of the Ba-sensitive
current studied using a voltage step paradigm showed characteristic
inward rectification (Fig. 2B). The graph illustrates
the time-dependent decrease in the GIRK1-mediated conductance, whereas
the reversal potential of the current does not change. These results
indicate that the reduction in the current was caused by a decrease in
the conductance and was not a consequence of a reduction in
K
driving force caused by K
loading
of the oocyte.
The rate constant of the decay was independent of the
peak response obtained (Fig. 2C). The peak response
could be varied by either changing the concentration of DAMGO used to
elicit the response (as in Fig. 2C) or by varying the
amount of receptor cRNA injected per oocyte. Additionally, oocytes from
different harvests showed widely differing peak responses to a
particular combination of DAMGO concentration and RNA amount injected.
To facilitate the comparison of the desensitization kinetics between
oocytes, the Ba-sensitive component of the current
(I
) for each oocyte was normalized against the peak
Ba
-sensitive component of the response
(I
) for that oocyte. Fig. 2D depicts the normalized current decay exponential for a control
population of oocytes that was obtained by averaging the normalized
decay exponentials obtained from each oocyte of the population. The
rate constant for this population of oocytes was 14.6 min.
ROMK1 is
another member of the family of cloned inwardly rectifying K channels but is not G protein-gated(23) . The inward
current generated in response to perfusion of hK in oocytes injected
with ROMK1 cRNA was not increased by mu receptor activation (data not
shown), and the current evoked in hK did not decay by more than 20% in
20 min (Fig. 2E). Thus, the desensitization shown by
GIRK1 is not a general property of inwardly rectifying potassium
channels expressed in this experimental system.
The 5HT1A receptor
and the GIRK1 channel have previously been shown to couple in Xenopus oocytes(1) . Prolonged exposure of oocytes
expressing 5HT1A receptors and GIRK1 to 8-OH-DPAT, a selective 5HT1A
receptor agonist, also caused a decay in the evoked response (Fig. 3A). The rate of decay in response to the
activation of the GIRK1 channel by the mu receptor was not
significantly different from the rate of decay in response to 8-OH-DPAT
in oocytes expressing the 5HT1A receptor (15.2 ± 1.8 min (n = 3) compared to 16.4 ± 2 min (n =
3), respectively). In oocytes coinjected with mu receptor, 5HT1A
receptor, and GIRK1, application of either agonist produced
cross-desensitization to the other (Fig. 3A). Under the
conditions chosen, stimulation with either 1 µM 8-OH-DPAT
or 1 µM DAMGO produced similar peak responses. As shown in
the upper trace in Fig. 3A, after the response
to 1 µM DAMGO had peaked, the superfusion was continued
for at least 20 min to allow desensitization. Subsequent application of
1 µM 8-OH-DPAT produced an attenuated response (I, upper panel, Fig. 3A) compared to an acute response to 8-OH-DPAT (I
, Fig. 3A, lower
panel). Fig. 3B demonstrates that desensitization
of the response obtained by the continued application of the mu opioid
agonist produced an equivalent attenuation of the response to the
activation of the 5HT1A receptor. The converse was also observed;
desensitization of the response by prior activation of the 5HT1A
receptor resulted in an equivalent attenuation of the response to the
activation of the mu opioid receptor. Application of 1 µM 8-OH-DPAT did not activate the potassium conductance in oocytes
expressing mu receptor and GIRK1 alone, and the same concentration of
DAMGO did not change the potassium conductance in oocytes injected with
5HT1A receptor and GIRK1 cRNA alone. The heterologous desensitization
observed indicates that desensitization occurred at a common component
following receptor activation.
Figure 3: Heterologous desensitization of the evoked current response through the stimulation of the 5HT1A and the mu opioid receptors. Oocytes were injected with cRNAs for the 5HT1A receptor, mu opioid receptor, and GIRK1 channel. A, a representative current trace obtained when an oocyte was exposed to 1 µM 8-OH-DPAT after the response evoked by 1 µM DAMGO had desensitized (upper panel). The peak response to 1 µM 8-OH-DPAT in an oocyte from the same batch is seen in the lower trace. The response to 1 µM DAMGO in this oocyte after the current response to 8-OH-DPAT has desensitized is much smaller than the peak response to the direct exposure to DAMGO (compare to upper trace). B, data from A presented as mean from three separate responses ± S.E. Each column represents the mean of the amplitude measures illustrated in A.
The desensitization of the evoked
current was further studied using GTPS, a nonhydrolyzable GTP
analog that maintains the G protein in an activated state (i.e.
-GTP and free
). Oocyte injection of GTP
S
increased I
by more than 100% whereas there was
no significant effect on the amplitude of the I
(Fig. 4A). The increase in the basal current
through the GIRK1 channels induced by GTP
S increased the total
inward current through these channels after application of DAMGO
(I
) (Fig. 4A). The
increase in the basal activity of the GIRK1 channel upon injection of
GTP
S suggests that a low level of constitutive activity of the G
protein may be partly responsible for the basal conductance observed.
It is notable that GTP
S had no effect on the time constant of
decay of the response (Fig. 4B). The lack of change in
the rate suggests that desensitization was not caused by a reduction in
the rate of G protein activation and the concentration of activated
G
or free
complex.
Figure 4:
Effect of GTPS on the current
responses in oocytes injected with mu opioid receptor and GIRK1
channel. Twenty-five nl of 40 mM GTP
S dissolved in 10
mM Tris (pH 7.5), or Tris buffer alone as control, were
injected into oocytes expressing the mu receptor and the GIRK1 channel. A, comparison between the amplitudes of the currents measured
in oocytes injected either with GTP
S (hatched columns) or
vehicle (filled columns). Measures of the Basal, Activated, and Bas.+Act. currents were made as
illustrated in Fig. 1A. B, the averaged,
normalized decay curve for a population of oocytes injected with
GTP
S compared with a population injected with buffer alone. In
this particular experiment the desensitization was not followed for
longer than 10 min as large leak currents appeared in oocytes injected
with GTP
S. All measures are expressed as means ± S.E. (n = 3).
In the absence of
activated receptor, oocytes expressing the GIRK1 channel alone showed a
basal level of activity as evident by the
Ba-sensitive current (I
) during
perfusion of hK. The basal current itself desensitized even in the
absence of receptor or receptor activation (Fig. 5A).
The rate of decay of this basal current was not significantly different
from the rate of decay after mu receptor activation (Fig. 5A).
Figure 5:
The rate of densensitization was
independent of receptor activation and not affected by an alternative
route of channel activation. A, oocytes were each injected
with either 1 ng of GIRK1 cRNA alone or 1 ng each of GIRK1 and mu
opioid receptor cRNA. In oocytes expressing the channel alone, an
inward GIRK1 current was evoked by superfusion in hK that attenuated
with time. In oocytes expressing both GIRK1 and mu receptors, an inward
GIRK1 current was evoked by application of 1 µM DAMGO in
hK (as illustrated in Fig. 2A). The rates of decay in
the amplitudes of the two responses (calculated as shown in Fig. 2D) were not significantly different. B,
a second group of oocytes was each injected with either 1 ng each of
GIRK1 and mu opioid receptor cRNA or with 1 ng each of GIRK1,
-adrenergic receptor, and G
cRNA. In
oocytes expressing both GIRK1 and mu receptors, an inward GIRK1 current
was evoked by application of 1 µM DAMGO in hK. In oocytes
expressing GIRK1,
-adrenergic receptor,
G
, responses were generated by application of 1
µM isoproterenol in hK. The rates of decay in the
amplitudes of the two responses were again not significantly
different.
In addition to activation by
G/G
-coupled receptors, GIRK1 can also be
activated by the
-adrenergic receptor but only in
oocytes also coinjected with cRNA for G
. (
)Activation of the
-adrenergic receptor by
isoproterenol evoked a GIRK1-mediated response that decayed by the same
kinetics as following mu receptor activation (Fig. 5B).
Thus, modification of the coupling pathway did not significantly affect
the desensitization process.
Since the desensitization response
observed thus far had followed the elevation of K concentration (inward currents were generated by switching the
buffer from ND96 to hK), we determined if desensitization could also be
observed if the oocytes were treated with agonist in ND96. Activation
of mu receptors by 1 µM DAMGO in oocytes coexpressing both
mu receptors and GIRK1, and bathed in ND96, produced an increase in
GIRK1-mediated conductance as determined from voltage steps performed (Fig. 6). Continuous application of the agonist in ND96 also
resulted in a decay of the conductance equivalent to that observed in
hK.
Figure 6:
DAMGO activation of GIRK1-mediated
conductances of oocytes bathed in ND96 solution. Oocytes expressing
both the mu receptor and GIRK1 channel were held at -80 mV in
ND96, then exposed to 1 µM DAMGO in ND96 for about 20 min
followed by the additional application of 300 µM Ba (in ND96). I-V relationships generated by
steps of 20 mV from -160 to 40 mV were derived for the
GIRK1-mediated conductance after the subtraction of the current, at the
same potential, in the presence of 300 µM Ba
. The GIRK1-mediated conductance of the oocyte
in ND96 (1) increased upon 2 min application of DAMGO (2) followed by a decay in conductance (3) after 20
min of DAMGO exposure. The figure shows the results from a
representative cell, and results were replicated three
times.
To identify factors that could significantly alter the
desensitization process, a series of treatments was tested (Table 1). Switching of buffer from ND96 to hK collapses the
Na gradient across the oocyte membrane which could in
turn lead to Ca
loading of the cell caused by an
inhibition of the Na
/Ca
exchanger(27) . The hK solution was modified to remove
Ca
and add 1 mM EGTA. The concentration of
Mg
was also increased to 4 mM to stabilize
the oocyte membrane in the absence of Ca
. In another
experiment the oocytes were preincubated for 20-30 min with
0.5-1 mM of BAPTA-AM, a membrane-permeable
Ca
chelator. BAPTA-AM treatment did effectively block
the Ca
-dependent Cl
current
activated by membrane depolarization (data not shown). Block of
Ca
-dependent mechanisms by BAPTA-AM or removal of
Ca
failed to alter the rate of current decay (Table 1). These results indicate that desensitization was not
Ca
-dependent.
cAMP-dependent kinase was stimulated by the application of combination of 0.5 mM 8-CPT-cAMP, 2 µM forskolin, and 0.5 mM IBMX, agents that will increase intracellular concentrations of cAMP. This preparation was shown to greatly increase the membrane conductance of oocytes injected with cRNA for the CFTR chloride channel (data not shown). Protein kinase C was stimulated by the application of 1 µM 12,13-phorbol didecanoate, a potent and specific activator. Protein kinases were nonspecifically inhibited by 1 µM staurosporine. Serine and threonine phosphatases were inhibited by 1 µM okadaic acid. None of these treatments altered the decay exponential (Table 1). Cytochalasin B (100 nM), which disrupts the cellular cytoskeletal machinery by binding to actin filaments, did not change the rate constant of decay. Thus, desensitization did not require internalization of components of the signal transduction machinery.
Chen and Yu(4) , reported that in Xenopus oocytes, the coupling of the mu opioid receptor to the GIRK1 channel was regulated by both protein kinase C and protein kinase A. They showed that treatment with 8-CPT-cAMP or the injection of the catalytic subunit of cAMP-dependent protein kinase abolished the desensitization of the response to DAMGO and that treatment with PMA enhanced the desensitization. Using the same protocol, we failed to replicate their findings (Fig. 7). Under the conditions used, a second exposure to 1 µM DAMGO produced a response that was approximately 20% less than the initial amplitude (Fig. 7A). Treatment of the oocytes with 1 mM 8-CPT-cAMP in ND96 between the two measurements had no effect on the amplitude of the second DAMGO-evoked response (Fig. 7B). Thus, activation of cAMP-dependent protein kinase had no effect on the desensitization process measured by this protocol. The basis for the discrepancy in findings is not known.
Figure 7: Action of 8-CPT-cAMP on response desensitization. Replication of experimental protocol described by Chen and Yu. Oocytes were injected with 1 ng of cRNA for both the mu receptor and the GIRK1 channel. A, a representative current trace from an oocyte illustrating the experimental protocol used. After the first DAMGO stimulation, the superfusate was switched to ND96, and the oocyte was treated with 8-CPT-cAMP (1 mM) for 10 min or with ND96 alone (as in this example). The superfusate was then switched back to hK solution to record the second DAMGO-induced membrane current. B, the second DAMGO-stimulated peak response was expressed as a percentage of the initial response to DAMGO. Treatment with 8-CPT-cAMP did not significantly affect the reduction the amplitude of the second DAMGO response. Data presented are means ± S.E.
We demonstrate that sustained agonist-induced activation of
GIRK1 leads to an increase in membrane conductance that subsequently
decays. The principal finding of this study is that the desensitization
of response is a result of events occurring downstream of the activated
neurotransmitter receptor and is probably a consequence of inactivation
of the GIRK1 channel. The heterologous desensitization observed with
the coinjected mu and 5HT1A receptor indicated that a common
post-receptor event, rather than a receptor-dependent process was
responsible. Furthermore, a receptor-independent mechanism for the
desensitization of the response was suggested by the finding that
injection of GTPS failed to alter the rate of response
desensitization. Evidence for channel inactivation was also provided by
the receptor-independent decay of basal channel activity upon perfusion
with hK.
Previous studies have established that the coupling of the
GIRK1 channel to the receptor is membrane delimited, does not occur
through diffusible cytosolic messengers, and is thought to be mediated
by the direct binding of activated G proteins to the channel (2, 3, and
see (5, 6, 7) ). Thus, the findings with
GTPS injection indicate that the desensitization occurred either
at the level of the G protein or through an inactivation of the channel
itself. The elevation in external K
concentration
which led to the desensitization of the basal conductance without
membrane depolarization or increase in internal Ca
(Fig. 5A) was more likely to have affected the
potassium channel directly than to have caused an inactivation of the G
proteins. The inwardly rectifying K
channel has been
shown to have a long pore with multiple binding sites for permeant
ions(28, 29, 30) . Our hypothesis is that
K
ions binding within the pore of the activated
channel induce a conformational change that allows its inactivation.
The desensitization of the mu opioid receptor response reconstituted
in oocytes may closely parallel a process occurring in vivo.
Harris and Williams (31) showed that the mu opioid-induced
activation of the inwardly rectifying potassium channel in locus
coeruleus neurons also acutely desensitizes. A large proportion of the
desensitization was homologous; however, a significant degree of
heterologous desensitization to
-adrenoreceptor-mediated hyperpolarization of the same
neurons was also observed(31) . This result revealed that some
desensitization of a common component of the signaling pathway also
occurred in the locus coeruleus neuron. In agreement with our findings,
the heterologous desensitization to the
-adrenoreceptor-mediated hyperpolarization was
unaffected by agents that alter kinase activity. Thus, although
tolerance to opioids results from multiple
mechanisms(14, 21) , our results may describe one,
physiologically relevant component.
A similar desensitization
process for acetylcholine regulation of the inward rectifier has been
described in mammalian myocytes which express a single muscarinic
receptor subtype(32) . Desensitization of this muscarinic
acetylcholine receptor induced increase of K conductance in cardiac purkinje fibers occurs over a similar time
period(33) ; these fibers, however, do not show a concomitant
desensitization of the process by which acetylcholine inhibits the
catecholamine-induced increase of slow inward current. This distinction
indicates that the desensitization was a result of a change in a
post-receptor component of the signaling pathway. Receptor-independent
desensitization of the cardiac G protein-gated K
conductance was also demonstrated by Kurachi et
al.(34) . The acetylcholine-induced K
channel current acutely desensitized in GTP
S-loaded atrial
cells. In addition, prior channel activation through P1-purinergic
receptors induced heterologous desensitization to subsequent
acetylcholine application. Thus, the desensitization of the GIRK1
channel response we demonstrated in Xenopus oocytes closely
resembles that described in cardiac cells(33, 34) .
Our findings suggest that the desensitization phenomena reported
previously in heart and locus coeruleus may have resulted from channel
inactivation.
Chen and Yu (4) demonstrate that repeated
stimulation of the mu receptor coinjected with the GIRK1 channel in Xenopus oocytes leads to desensitization of the mu-activated
current response. They observed an effect of protein kinase A and
protein kinase C on the amplitude of the second mu-activated response.
Previous reports have established that phosphorylation is not involved
in the coupling of neurotransmitter receptors to the G protein-gated
inward rectifier K channel (see (5) and (6) ). In agreement with the literature and in contrast to the
findings presented in (4) , we observed no actions of agents
that alter the phosphorylation state of cellular proteins, on either
the rate of response desensitization or on the amplitudes of the basal
GIRK1 or the mu-activated currents.
The concept that desensitization may result from a change in the ion channel has precedent. Several ligand-gated ion channels including the nicotinic acetylcholine receptor exist at equilibrium in two predominant conformations, the resting and the desensitized states. Prolonged exposure to several agonists shifts the affinity toward the desensitized state in a slow reaction (100 ms to 1 min) and this occurs in the absence of any covalent modifications(26) . Thus, Xenopus oocytes provide an accessible system to study the mechanisms of desensitization following prolonged opioid agonist exposure and may elucidate the role of GIRK1 channel desensitization in tolerance phenomena in vivo.