©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Agonist-induced Desensitization of the Mu Opioid Receptor-coupled Potassium Channel (GIRK1) (*)

(Received for publication, September 8, 1994; and in revised form, October 27, 1994)

Abraham Kovoor (1) Douglas J. Henry (1) Charles Chavkin (1) (2)(§)

From the  (1)Department of Pharmacology, University of Washington, Seattle, Washington 98195 and the (2)Computational Neuroscience Program, Caltech, Pasadena, California 91125

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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^2,MePhe^4,Glyol^5]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.


INTRODUCTION

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 beta 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, (^1)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.


MATERIALS AND METHODS

Chemicals

DAMGO was from Peninsula Laboratories. Naloxone was from Research Biochemicals International. BAPTA-AM was from Calbiochem. All other chemicals were from Sigma. GIRK1 channel and 5HT1A cRNA were prepared as described(3) ; cRNA for G and beta(2)-adrenergic receptor were provided by Dr. Nancy Lim (Caltech). cRNA for ROMK1 was similarly prepared from a clone provided by Dr. Lily Jan(23) . The rat mu opioid receptor clone was provided by Dr. Lei Yu(24) . The amount of mu receptor cDNA was first increased by the utilization of Amplitaq DNA Polymerase (Perkin Elmer) in a standard polymerase chain reaction using oligonucleotides designed to add an SP6 promoter region and a 45-base poly(A) tail. Purity and yield of the product was verified by gel electrophoresis and the measurement of absorbance spectra. SP6 RNA Polymerase (Ambion Corp.) was used to generate capped cRNA. The cRNA was then dissolved in RNase-free water and stored at -75 °C until oocyte injection.

Oocyte Culture and Injection

Oocytes were prepared as described(25) . The oocytes were incubated for 3-7 days after injection of the cRNAs in ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), and 5 mM HEPES, pH 7.5) solution supplemented with sodium pyruvate (2.5 mM), gentamycin (50 µg/ml), and 5% (v/v) heat inactivated horse serum (Life Technologies, Inc.). cRNA was injected into oocytes with a Drummond microinjector (maximum volume injected was <50 nl/oocyte).

Electrophysiology

Oocytes were voltage clamped at -80 mV with two electrodes filled with 3 M potassium chloride and with resistances of 0.5-1.5 M, using an Axoclamp-2A and pCLAMP software (Axon Instruments). Oocytes were superfused with either ND96 or high K (hK) solution which is identical to ND96 except that the concentrations of NaCl and KCl are reversed.


RESULTS

Coupling of the Mu Opioid Receptor to the GIRK1 Channel

To study the coupling of the mu opioid receptor to the GIRK1 channel, we expressed both proteins in Xenopus oocytes. Messenger RNAs for the two clones were generated by in vitro transcription, and oocytes were injected with either cRNA alone or with both cRNAs. Coupling was studied using two electrode voltage clamp. In one of the paradigms used, cells were clamped at -80 mV while bathed in normal ND96 solution containing 96 mM Na and 2 mM K. Cells were then superfused with a high K (hK) solution, with 96 mM K and 2 mM Na. In this solution the K equilibrium potential (E(K)) approaches 0 mV and enables inward current flow through inwardly rectifying K channels at negative holding potentials.

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(2) (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.



Desensitization of the Response Mediated by the GIRK1 Channel

A large inward current was observed when the superfusion buffer was switched from ND96 to hK containing 1 µM DAMGO. The evoked inward current attenuated with time (Fig. 2A). The current decay curve could be fit by a single exponential with a time constant of 14.3 min (r^2 = 0.982). To determine whether desensitization was a reversible process, we allowed the response to desensitize by exposure to 1 µM DAMGO in hK for 20 min. Oocytes were then perfused with ND96 alone for 1 h while clamped at -80 mV, and at which time the amplitude of the response to reapplication of 1 µM DAMGO in hK was the same as the initial peak response (data not shown).


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. alpha-GTP and free beta). Oocyte injection of GTPS 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 GTPS 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 GTPS 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 GTPS 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 beta 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 GTPS 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 GTPS (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 GTPS 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 GTPS. 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, beta(2)-adrenergic receptor, and GcRNA. 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, beta(2)-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(i)/G(o)-coupled receptors, GIRK1 can also be activated by the beta(2)-adrenergic receptor but only in oocytes also coinjected with cRNA for G. (^2)Activation of the beta(2)-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.




DISCUSSION

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 alpha(2)-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 alpha(2)-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 GTPS-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.


FOOTNOTES

*
This work was supported by an individual postdoctoral fellowship Grant DA 05160 (to D. J. H.) and Grant DA 04123 (to C. C.) from the National Institute on Drug Abuse. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, SJ 30, University of Washington Seattle, WA 98195. Tel.: 206-543-4266; Fax: 206-685-3822.

(^1)
The abbreviations used are: DAMGO, [D-Ala^2,MePhe^4,Glyol^5]enkephalin; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; 8-cPT-cAMP, 8-(4-chlorophenylthio)-cAMP; IBMX, 3-isobutyl-1-methylxanthine; GTPS, guanosine 5`-O-(thiotriphosphate); CFTR, cystic fibrosis transmembrane regulator; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`tetraacetic acid tetra-(acetoxymethyl)-ester.

(^2)
N. F. Lim, N. Dascal, C. Labarca, N. Davidson, and H. A. Lester, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Henry Lester for helpful discussion and cDNA clones, Dr. Lei Yu for the generous gift of the mu opioid receptor clone, and Dr. Nathan Dascal for sharing unpublished data and for helpful discussion.


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