Correspondence to: Gerry S. Oxford, Department of Cell & Molecular Physiology, CB # 7545, 452 MSRB, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Fax:919-966-6927 E-mail:gsox{at}med.unc.edu.
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Abstract |
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The human D3 dopamine receptor can activate G-proteincoupled inward rectifier potassium channels (GIRKs), inhibit P/Q-type calcium channels, and inhibit spontaneous secretory activity in AtT-20 neuroendocrine cells (Kuzhikandathil, E.V., W. Yu, and G.S. Oxford. 1998. Mol. Cell. Neurosci. 12:390402; Kuzhikandathil, E.V., and G.S. Oxford. 1999. J. Neurosci. 19:16981707). In this study, we evaluate the role of GIRKs in the D3 receptor-mediated inhibition of secretory activity in AtT-20 cells. The absence of selective blockers for GIRKs has precluded a direct test of the hypothesis that they play an important role in inhibiting secretory activity. However, the tetrameric structure of these channels provides a means of disrupting endogenous GIRK function using a dominant negative approach. To develop a dominant-negative GIRK mutant, the K+ selectivity amino acid sequence -GYG- in the putative pore domain of the human GIRK2 channels was mutated to -AAA-, -GLG-, or -GFG-. While the mutation of -GYG- to -GFG- did not affect channel function, both the -AAA- and -GLG- GIRK2 mutants were nonfunctional. This suggests that the aromatic ring of the tyrosine residue rather than its hydroxyl group is involved in maintaining the pore architecture of human GIRK2 channels. When expressed in AtT-20 cells, the nonfunctional AAA-GIRK2 and GLG-GIRK2 acted as effective dominant-negative mutants and significantly attenuated endogenous GIRK currents. Furthermore, these dominant-negative mutants interfered with the D3 receptor-mediated inhibition of secretion in AtT-20 cells, suggesting they are centrally involved in the signaling pathway of this secretory response. These results indicate that dominant-negative GIRK mutants are effective molecular tools to examine the role of GIRK channels in vivo.
Key Words: potassium channel structure, calcium channels, selectivity filter, autoreceptor, FM1-43
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INTRODUCTION |
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The molecular mechanisms underlying the dopaminergic regulation of mammalian neurotransmission are not well characterized. Among G proteincoupled dopamine receptors, the role of the D3 receptor in regulating secretory activity, in particular, is poorly understood. The D3 receptor has been proposed to function as an autoreceptor regulating the release of dopamine at nerve terminals (
In the AtT-20 expression system, we have demonstrated that D3 receptors can efficiently couple to two different ion-channel effectors. D3 receptors can activate endogenous G proteincoupled inward rectifier potassium channels (GIRKs) with high efficacy (
The GIRK subfamily consists of five major isoforms (GIRK1GIRK5 or Kir3.1Kir3.5). Functional GIRK channels are tetramers composed of either four identical (homomeric) or nonidentical (heteromeric) subunits. All known GIRKs form heteromultimers in vivo (
We have previously shown, using reverse transcriptase PCR and Western blot analysis, that AtT-20 cells express only GIRK1 and GIRK2 isoforms (
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MATERIALS AND METHODS |
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Generation of Mutant GIRK2 Constructs
The mutant GIRK2 constructs were generated by site-directed mutagenesis. The plasmid encoding the wild-type human GIRK2 gene was denatured and annealed to a selection primer (which converted a unique StuI restriction enzyme site in hGIRK2 to PpuMI) as well as the mutagenic primers (encoding either the GYG AAA, GYG
GLG, or GYG
GFG mutations). Second-strand DNA synthesis from these annealed primers was carried out using T4 DNA polymerase (New England Biolabs, Inc.). The gaps in the modified plasmids were sealed using T4 DNA ligase (New England Biolabs, Inc.). StuI restriction enzyme was used to linearize unmodified plasmids while not affecting the modified plasmids (which had incorporated the selection and mutagenic primers). This step reduced the subsequent transformation efficiency of linear unmodified plasmids compared with the circular modified plasmids. The mixture of linear unmodified and circular modified plasmids were then transformed into Escherichia coli BMH 71-18 mutS (CLONTECH Laboratories, Inc.). This strain, being DNA mismatch repair deficient, allows the propagation of modified plasmids containing the selection and mutagenic primer. Plasmid DNA isolated from transformed E. coli BMH 71-18 mutS colonies was pooled and redigested with StuI to linearize unmodified plasmids and further enrich the population of circular modified plasmids. This mixture of plasmids was then transformed into E. coli DH5a (GIBCO BRL). Plasmid DNA was isolated from individual colonies and characterized by both restriction enzyme mapping and DNA sequencing. Clones that contained the desired mutations in the hGIRK2 gene were identified and subjected to further DNA sequencing to confirm that this was the only mutation incorporated into the hGIRK2 gene.
Generation of GIRK2Enhanced Green Fluorescent Protein Fusion Constructs
Wild-type and mutant GIRK2 genes were fused in frame at the carboxyl terminal to the coding region of enhanced green fluorescent protein (EGFP) in the EGFP-N2 plasmid (CLONTECH Laboratories, Inc.). In brief, the carboxyl terminal region of the human GIRK2 constructs were amplified by PCR using an upper primer containing the unique BstEII restriction enzyme site and a lower primer that lacked the GIRK2 stop codon. The lower primer also incorporated a unique XmaI restriction enzyme site that allowed the in-frame introduction of the GIRK2 gene into the EGFP-N2 plasmid. The BstEII-XmaI PCR fragment was subcloned along with the remaining human GIRK2 sequence into the EGFP-N2 plasmid. As a result of the subcloning procedure, the recombinant GIRK2 fusion construct contains a linker region of nine amino acids (PGIHRPVAT) in between the terminal valine residue of human GIRK2 and the first methionine residue of EGFP.
Cell Culture
Chinese hamster ovary (CHO) cells were grown in Ham's F12 medium with 10% FCS and 10,000 U of penicillin/streptomycin. AtT-20 mouse pituitary cells were grown in Ham's F10 medium with 5% FBS, 20% heat-inactivated horse serum, 200 mM glutamine, and 1 mg/ml gentamicin. CHO and AtT-20 cells stably expressing human dopamine receptors were maintained in 200 mg/ml and 500 µg/ml of geneticin (G418), respectively. For transient transfections and subsequent electrophysiological characterization, cells were plated onto glass coverslips coated with 40 µg/ml poly L-lysine.
Transfection of Receptors and Channels into AtT-20 and CHO Cells
AtT-20 cells stably expressing the human D3 receptor were generated by clonal selection after a Pfx-2 reagent (Invitrogen Corp.)-mediated transfection. CHO-K1 cells stably expressing either the human D3 receptor (CHO-D3) or the human short isoform of the D2 receptor (CHO-D2S) were gifts from Dr. Tony Sandrasagra (Hoechst-Marion Roussel, Somerville, NJ). Transient-transfections into CHO cells were done using Lipofectamine (GIBCO BRL) and into AtT-20 cells using Pfx-2 (Invitrogen Corp.). To identify transfected cells, we used plasmids encoding either the EGFP (CLONTECH Laboratories, Inc.) or the CD4 membrane antigen (a gift from ICAgen Inc.). The latter marker was used in experiments with either fluo-3 or FM1-43 dyes (Molecular Probes, Inc.), since these compounds have excitation and emission wavelengths that overlap with EGFP. The cells expressing the CD4 membrane antigen were identified using Dynabeads® M-450 CD4 (Dynal). Transfection efficiency of 1530% was routinely achieved.
Electrophysiology
Agonist-activated currents were measured in AtT-20 or CHO cells in the whole-cell configuration of the patch clamp using an Axopatch 200 amplifier (Axon Instruments, Inc.). Patch pipettes were constructed from N51A glass (Drummond), coated with dental wax (Kerr Sticky Wax), and polished on a homemade microforge at 600x magnification. Currents were elicited by ramp voltage commands (-120 to +40 mV), followed by a hyperpolarizing step (-100 mV) from holding potentials of -60 mV. The current responses were normalized to the cell capacitance (picoamperes per picofarad), to account for variation in cell size. The standard external solution (SES) used contained (mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. The pipette solution contained (mM): 130 K-Aspartate, 20 NaCl, 10 HEPES, 10 glucose, 0.1 GTP, 5 Mg-ATP, 1 EGTA. To enhance inwardly rectifying K+ currents in the voltage-clamp experiments, controls and drug exposures were performed in solutions with elevated extracellular [K+] (50 mM) by substitution for Na+. Quinpirole and somatostatin (RBI Chemicals) were used at 100 nM concentration, unless otherwise indicated. Drug solutions were delivered to cells via a multibarreled micropipette array (Drummond Microcaps, 3 µl).
Data Acquisition and Analysis
Whole-cell macroscopic currents in response to ramp and step commands were sampled via a Digidata 1200b interface using Axotape and pClamp 7.0 software (Axon Instruments, Inc.). Data files are then imported into SigmaPlot for display or analysis.
Intracellular Calcium Imaging
The cells on glass coverslips were rinsed in PBS and incubated at 37°C in 5 mM fluo-3 AM (Molecular Probes, Inc.) for 30 min. Cells were rinsed in SES and placed in a glass-bottom chamber on an inverted microscope stage (Nikon). Drug and control solutions were directly applied using a continuous flow (~1 ml/min) bath system. After excitation at 485 nm, the fluorescence emission was band pass filtered at 535 nm, collected via a quartz phase objective (40x or 100x), amplified by a Videoscope KS-1381 intensifier, and passed to a Pentamax cooled CCD camera (Princeton Instruments). Video images were captured using the Metamorph software package (Universal Imaging Corp.). This software allows logging of fluorescence intensity versus time for several cells as a measure of intracellular [Ca2+] changes (arbitrary units).
Imaging Vesicle Trafficking
The imaging procedures employed have been described previously (
Statistical Methods
Student's t test was performed on relevant data using SigmaPlot (SPSS Inc.). In the t test, the data was considered statistically different when the probability value was <0.05.
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RESULTS |
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Generation and Characterization of Mutant GIRK2 Channels
To generate dominant-negative GIRK2 mutants, we mutated the -GYG- amino acid sequence in the putative pore region of the human GIRK2 channel to either GFG, GLG, or AAA (Fig 1). The mutants were generated by site-directed mutagenesis as described in MATERIALS AND METHODS. To determine whether the mutation affected expression and cellular localization, we also generated recombinant constructs in which both the wild-type and mutant GIRK2 proteins were fused in frame at the carboxyl terminal to the EGFP. These fusion constructs were individually transfected into CHO cells and their expression pattern determined by fluorescence microscopy. CHO cells transfected with the wild-type and mutant GIRK2::EGFP fusion constructs exhibit distinct organelle and membrane fluorescence staining patterns that are qualitatively different from the uniform fluorescence pattern observed in CHO cells transfected with control EGFP plasmid (data not shown). These results (and the functional studies below) suggest that both wild-type and mutant constructs are expressed on the surface of the cells and that mutation of the GYG sequence in the pore region does not detectably alter expression or localization of the GIRK2 channels.
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Functional Characterization of GIRK2 Mutants in CHO Cells
Having shown that the mutant GIRK2 subunits are expressed, we next determined whether the expression yielded functional channels. Wild-type and mutant GIRK2 channels (the non-EGFPfused constructs) were transfected individually into CHO cells stably expressing either the human D2S or human D3 receptor, along with EGFP marker constructs. Transfected cells were identified by EGFP expression and whole-cell voltage clamp recording was performed as described in MATERIALS AND METHODS. Fig 2 shows representative quinpirole-induced inwardly rectifying currents elicited by voltage ramps applied to separate CHO cells, each coexpressing the human D2S receptor and a different human GIRK2 construct as indicated. As mentioned previously, the nontagged constructs were routinely employed to avoid interference with calcium or vesicle cycling measurements using similar fluorescence wavelengths; however, we have confirmed that similar responses occur in cells expressing the EGFP fusion constructs of each isoform. These records suggest that the GFG-GIRK2 mutant can make functional homomultimeric channels that are qualitatively indistinguishable from wild type, whereas neither GLG- nor AAA-GIRK2 mutants yield agonist-induced currents and are presumably nonfunctional.
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In Fig 3, we have plotted the mean normalized current densities at -100 mV in SES, 50K-ES, or 100 nM quinpirole (in 50K-ES) for the wild-type and the three mutant GIRK2 channels. The data indicate that wild-type GIRK2 and GFG-GIRK2 yield both constitutive and quinpirole-induced currents in CHO cells expressing either the human D2S (Fig 3 A) or the human D3 (B) receptors. In contrast, the GLG-GIRK2 and AAA-GIRK2 mutants do not generate either constitutive or quinpirole-induced currents. Similar results were obtained with the GIRK2-EGFP fusion constructs shown in Fig 1, suggesting that the carboxyl-terminal EGFP fusion does not affect function of these channels (data not shown). The remaining experiments in this report employed the non-EGFPfused constructs for consistency.
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The results of Fig 2 and Fig 3 indicate that while the mutation of the GYG sequence to GFG does not affect channel function, mutation to either GLG or AAA renders the human GIRK2 channel nonfunctional when expressed as a homomultimer.
The GLG- and AAA-GIRK2 Constructs Function as Dominant-Negative Mutants
To determine whether the GLG- and AAA-GIRK2 constructs could serve as dominant-negative elements, we assessed the ability of these mutants to disrupt the function of endogenous GIRK channels in AtT-20 mouse pituitary cells. We have previously shown that AtT-20 cells express only GIRK1 and GIRK2 isoforms and that the native channels couple to endogenous somatostatin receptors or transfected human D3 receptors (
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Dominant-Negative GIRK2 Mutants Alter the Ability of D3 Receptors to Inhibit Spontaneous Action Potentials in AtT-20 Cells
AtT-20 cells are excitable and fire spontaneous action potentials (
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AAA-GIRK2 Mutant Disrupts the Ability of D3 Receptor to Inhibit Spontaneous Calcium Influx
Using the calcium indicator dye Fluo-3, we demonstrated that the spontaneous action potentials in AtT-20 cells are accompanied by a calcium influx that can also be inhibited by activation of either endogenous somatostatin receptors or heterologously expressed D3 receptors () are shown in Fig 6 A. In the cell expressing GFG-GIRK2, the intracellular calcium level is elevated at the beginning of the recording, presumably coinciding with the appearance of spontaneous electrical activity. Upon application of quinpirole, the calcium levels decline reversibly, as expected for a hyperpolarizing response that suppresses calcium-dependent action potential activity (e.g., Fig 5A and Fig B). In contrast, in the cell expressing the AAA mutant, application of the agonist failed to reduce calcium levels. The results of all such observations are summarized in Fig 6 B, where intracellular calcium levels are reduced on average by 45% by the activation of the human D3 receptor in GFG-GIRK2transfected cellsa reduction comparable with that seen in untransfected cells (see also
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AAA-GIRK2 Mutants Disrupt the Ability of D3 Receptors to Inhibit Spontaneous Secretory Activity in AtT-20 Cells
AtT-20 cells secrete ACTH and acetylcholine (). In AtT-20 cells transfected with the GFG-GIRK2 mutant, both the staining and destaining of FM1-43 fluorescence in the presence of 100 nM quinpirole are significantly inhibited compared with SES. The fluorescence intensity during the destaining process in SES or 100 nM quinpirole was normalized and replotted on a semilogarithmic scale and fit to a regression line for kinetic comparison (Fig 7 A, inset). It is apparent that the destaining process was dramatically retarded by D3 receptor activation. Fig 7 B shows cumulative destaining rates obtained from the slope of the regression line, indicating a significant decrease in the presence of 100 nM quinpirole. These results are essentially identical to our previous observations in untransfected AtT-20 cells stably expressing the human D3 receptor (
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DISCUSSION |
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The present study provides novel evidence that GIRK2 channels play an important role in mediating D3 receptor modulation of secretory activity. The D3 dopamine receptor has been suggested to modulate dopamine release at nerve terminals functioning as inhibitory autoreceptors in dopaminergic neurons (
The concept that activation of a potassium channel, rather than direct inhibition of a calcium channel, could be the major mechanism linking inhibitory neurotransmitters to reduction of secretion in neural and endocrine has been proposed previously. In the case of regulation of prolactin secretion by D2 receptor activation in pituitary lactotrophs, where direct calcium channel inhibition does not occur (2A adrenergic receptor. They found that a mutation of the
2A receptor (D79N) previously shown to block coupling of the receptor to GIRK channels, but not to calcium channels, could blunt agonist-induced inhibition of ACTH secretion when expressed in AtT20 cells, suggesting a role for GIRK channels in the action of these exogenous receptors. Finally, a link between somatostatin receptors and potassium channels was found to underlie both hyperpolarization and suppression of calcium levels in GH4 cells (
While this study represents the first use of a dominant-negative approach to study GIRK function, one previous study has reported the over expression of wild-type GIRK channels in cultured hippocampal neurons using an adenovirus gene transfer method (
Mice exhibiting the weaver phenotype have a G156S mutation in the GIRK2 gene (
The results from this study also raise some interesting questions about the role of certain amino acid residues of the selectivity filter in maintaining the pore architecture of GIRK2 channels. Based on the recent bacterial potassium channel crystal structure,
In conclusion, this study has identified a role for GIRK channels in mediating the inhibition of secretory activity by the D3 receptor. While the D3 receptor can directly inhibit voltage-gated calcium channels, it appears that the coupling of the D3 receptor to GIRK channels primarily underlies the inhibition of spontaneous secretory activity in AtT-20 cells. The mutational studies presented in this report have also provided some insight into the role of the tyrosine residue in the selectivity filter region of human GIRK2-containing channels. Furthermore, given the lack of selective blockers for GIRK channels, the study shows that the dominant negative approach can be used to selectively ablate GIRK function in vivo, thereby facilitating the study of their role in neuronal signaling.
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Footnotes |
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1 Abbreviations used in this paper: ACTH, adrenocorticotropic hormone; CHO cell, Chinese hamster ovary cell; EGFP, enhanced green fluorescent protein; GIRK channel, G-proteincoupled inward rectifier potassium channel; SES, standard external solution.
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Acknowledgements |
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We express our sincere appreciation to Rakhshi Khan for assistance in generating the dominant-negative mutants.
This work was supported by grants from the National Institutes of Health (NS18788) to G.S. Oxford and a Howard Hughes Medical Institute Pilot Studies grant to E.V. Kuzhikandathil.
Submitted: 18 January 2000
Revised: 20 March 2000
Accepted: 17 April 2000
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References |
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