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Dominant-negative mutants identify a role for GIRK channels in D3 dopamine receptor-mediated regulation of spontaneous secretory activity.

Kuzhikandathil EV, Oxford GS - J. Gen. Physiol. (2000)

Bottom Line: While the mutation of -GYG- to -GFG- did not affect channel function, both the -AAA- and -GLG- GIRK2 mutants were nonfunctional.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.These results indicate that dominant-negative GIRK mutants are effective molecular tools to examine the role of GIRK channels in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 27599, USA.

ABSTRACT
The human D3 dopamine receptor can activate G-protein-coupled 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. 12:390-402; Kuzhikandathil, E.V., and G.S. Oxford. 1999. J. Neurosci. 19:1698-1707). 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.

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Functional characterization of GIRK2 mutants in CHO cells. Bars represent mean (±SEM) current densities at −100 mV from CHO cells transfected with the GIRK2 constructs and either the human D2S dopamine receptor (A) or the human D3 dopamine receptor (B). Wild-type and mutant GIRK2 constructs (non–EGFP-tagged forms) were cotransfected with a 10-fold excess of dopamine receptors. Whole-cell voltage-clamp recording was done 24–36 h after transfection. The numbers in parenthesis represent the number of cells tested in each case. The GLG and AAA mutants exhibited significantly reduced inward currents compared with either wild type (WT) or GFG human GIRK2 in an external solution containing either 50 mM potassium (50 K-ES, hatched bars) or dopamine receptor agonist quinpirole (100 nM QP, cross-hatched bars) (*,#P < 0.01, Student's t test).
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Figure 3: Functional characterization of GIRK2 mutants in CHO cells. Bars represent mean (±SEM) current densities at −100 mV from CHO cells transfected with the GIRK2 constructs and either the human D2S dopamine receptor (A) or the human D3 dopamine receptor (B). Wild-type and mutant GIRK2 constructs (non–EGFP-tagged forms) were cotransfected with a 10-fold excess of dopamine receptors. Whole-cell voltage-clamp recording was done 24–36 h after transfection. The numbers in parenthesis represent the number of cells tested in each case. The GLG and AAA mutants exhibited significantly reduced inward currents compared with either wild type (WT) or GFG human GIRK2 in an external solution containing either 50 mM potassium (50 K-ES, hatched bars) or dopamine receptor agonist quinpirole (100 nM QP, cross-hatched bars) (*,#P < 0.01, Student's t test).

Mentions: 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-EGFP–fused constructs for consistency.


Dominant-negative mutants identify a role for GIRK channels in D3 dopamine receptor-mediated regulation of spontaneous secretory activity.

Kuzhikandathil EV, Oxford GS - J. Gen. Physiol. (2000)

Functional characterization of GIRK2 mutants in CHO cells. Bars represent mean (±SEM) current densities at −100 mV from CHO cells transfected with the GIRK2 constructs and either the human D2S dopamine receptor (A) or the human D3 dopamine receptor (B). Wild-type and mutant GIRK2 constructs (non–EGFP-tagged forms) were cotransfected with a 10-fold excess of dopamine receptors. Whole-cell voltage-clamp recording was done 24–36 h after transfection. The numbers in parenthesis represent the number of cells tested in each case. The GLG and AAA mutants exhibited significantly reduced inward currents compared with either wild type (WT) or GFG human GIRK2 in an external solution containing either 50 mM potassium (50 K-ES, hatched bars) or dopamine receptor agonist quinpirole (100 nM QP, cross-hatched bars) (*,#P < 0.01, Student's t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2232885&req=5

Figure 3: Functional characterization of GIRK2 mutants in CHO cells. Bars represent mean (±SEM) current densities at −100 mV from CHO cells transfected with the GIRK2 constructs and either the human D2S dopamine receptor (A) or the human D3 dopamine receptor (B). Wild-type and mutant GIRK2 constructs (non–EGFP-tagged forms) were cotransfected with a 10-fold excess of dopamine receptors. Whole-cell voltage-clamp recording was done 24–36 h after transfection. The numbers in parenthesis represent the number of cells tested in each case. The GLG and AAA mutants exhibited significantly reduced inward currents compared with either wild type (WT) or GFG human GIRK2 in an external solution containing either 50 mM potassium (50 K-ES, hatched bars) or dopamine receptor agonist quinpirole (100 nM QP, cross-hatched bars) (*,#P < 0.01, Student's t test).
Mentions: 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-EGFP–fused constructs for consistency.

Bottom Line: While the mutation of -GYG- to -GFG- did not affect channel function, both the -AAA- and -GLG- GIRK2 mutants were nonfunctional.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.These results indicate that dominant-negative GIRK mutants are effective molecular tools to examine the role of GIRK channels in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 27599, USA.

ABSTRACT
The human D3 dopamine receptor can activate G-protein-coupled 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. 12:390-402; Kuzhikandathil, E.V., and G.S. Oxford. 1999. J. Neurosci. 19:1698-1707). 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.

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Related in: MedlinePlus