scholarly journals G protein–coupled receptors differentially regulate glycosylation and activity of the inwardly rectifying potassium channel Kir7.1

2018 ◽  
Vol 293 (46) ◽  
pp. 17739-17753 ◽  
Author(s):  
Sheridan J. Carrington ◽  
Ciria C. Hernandez ◽  
Daniel R. Swale ◽  
Oluwatosin A. Aluko ◽  
Jerod S. Denton ◽  
...  
Keyword(s):  
Potassium Channel ◽  
G Protein ◽  
Single Channel ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
G Protein Coupled Receptors ◽  
Open Probability ◽  
Inwardly Rectifying Potassium Channel ◽  
Inwardly Rectifying ◽  
G Protein Coupled

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein–coupled inwardly rectifying potassium (GIRK) channels by G protein–coupled receptors (GPCRs) via the G protein βγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1.

scholarly journals The epithelial potassium channel Kir7.1 is stimulated by progesterone

2021 ◽  
Vol 153 (10) ◽  
Author(s):  
Ida Björkgren ◽  
Sarah Mendoza ◽  
Dong Hwa Chung ◽  
Monika Haoui ◽  
Natalie True Petersen ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Progesterone Receptors ◽  
Current Clamp ◽  
Inwardly Rectifying Potassium Channel ◽  
Whole Cell Patch Clamp ◽  
Inwardly Rectifying ◽  
The Brain ◽  
Precise Molecular Mechanism

The choroid plexus (CP) epithelium secretes cerebrospinal fluid and plays an important role in healthy homeostasis of the brain. CP function can be influenced by sex steroid hormones; however, the precise molecular mechanism of such regulation is not well understood. Here, using whole-cell patch-clamp recordings from male and female murine CP cells, we show that application of progesterone resulted in specific and strong potentiation of the inwardly rectifying potassium channel Kir7.1, an essential protein that is expressed in CP and is required for survival. The potentiation was progesterone specific and independent of other known progesterone receptors expressed in CP. This effect was recapitulated with recombinant Kir7.1, as well as with endogenous Kir7.1 expressed in the retinal pigment epithelium. Current-clamp studies further showed a progesterone-induced hyperpolarization of CP cells. Our results provide evidence of a progesterone-driven control of tissues in which Kir7.1 is present.

Single-channel properties of a G-protein-coupled inward rectifier potassium channel in brain neurons

1996 ◽  
Vol 75 (1) ◽  
pp. 318-328 ◽  
Author(s):  
J. J. Grigg ◽  
T. Kozasa ◽  
Y. Nakajima ◽  
S. Nakajima
Keyword(s):  
G Protein ◽  
Single Channel ◽  
Inward Rectifier ◽  
Equilibrium Potential ◽  
K Channel ◽  
Open Probability ◽  
K Concentration ◽  
Inwardly Rectifying ◽  
G Protein Coupled ◽  
Cytoplasmic Side

1. In cultured rat locus coeruleus neurons, somatostatin or met-enkephalin induces an inwardly rectifying K+ conductance. This inward rectifier was analyzed at the single-channel level. 2. Using the inside-out patch-clamp, guanosine 5'-triphosphate (GTP) application to the cytoplasmic side in the presence of somatostatin or met-enkephalin in the pipette produced a large increase in channel activity, which disappeared on switching from GTP to guanosine 5'-diphosphate. 3. The unitary conductance was approximately 30 pS at -95 mV with an extracellular K+ concentration of 156 mM and an intracellular K+ concentration of 124 mM at 23 degrees C. The channel showed burst behavior, and the closed time histogram was fit by two exponentials, with the fast time constant being 0.4 ms. The burst time histogram was also fit by two exponentials, with time constants of 0.24 and 2.0 ms (at 10 nM somatostatin). When the somatostatin concentration was changed from 500 to 1 nM, the kinetic behavior of the channel did not change, except that the open probability of the patch was decreased. 4. The current-voltage relation of the unitary channel current showed inward rectification. The reversal potential coincided with the K+ equilibrium potential, and it shifted according to a change in the K+ equilibrium potential. 5. In the presence of external somatostatin, the application of guanosine 5'-O-(3-thiotriphosphate) to the cytoplasmic side induced an irreversible activation of this channel. 6. These results indicate that this K+ channel is the microscopic counterpart of the somatostatin- or met-enkephalin-induced inwardly rectifying K+ current in whole cell recording, and that the channel is activated by a G protein without a diffusible second messenger. Thus this channel is identified as a neuronal G-protein-coupled inward rectifier K+ channel. 7. Analysis of the burst behavior, based on a close-close-open kinetic model, revealed that there are at least four states in the K+ channel, a short gap, a longer closing, a short opening, and a long opening, and that the neuronal inward rectifier is activated at faster rates than the atrial inward rectifier.

scholarly journals The inhibitory effect of Gβγ and Gβ isoform specificity on ENaC activity

2013 ◽  
Vol 305 (9) ◽  
pp. F1365-F1373 ◽  
Author(s):  
Ling Yu ◽  
Otor Al-Khalili ◽  
Billie Jeanne Duke ◽  
James D. Stockand ◽  
Douglas C. Eaton ◽  
...  
Keyword(s):  
Single Channel ◽  
Inhibitory Effect ◽  
G Protein Coupled Receptors ◽  
Open Probability ◽  
A6 Cells ◽  
Kinase C ◽  
Downstream Effectors ◽  
Isoform Specificity ◽  
Low Levels ◽  
G Protein Coupled

Epithelial Na+ channel (ENaC) activity, which determines the rate of renal Na+ reabsorption, can be regulated by G protein-coupled receptors. Regulation of ENaC by Gα-mediated downstream effectors has been studied extensively, but the effect of Gβγ dimers on ENaC is unclear. A6 cells endogenously contain high levels of Gβ1 but low levels of Gβ3, Gβ4, and Gβ5 were detected by Q-PCR. We tested Gγ2 combined individually with Gβ1 through Gβ5 expressed in A6 cells, after which we recorded single-channel ENaC activity. Among the five β and γ2 combinations, β1γ2 strongly inhibits ENaC activity by reducing both ENaC channel number ( N) and open probability ( Po) compared with control cells. In contrast, the other four β-isoforms combined with γ2 have no significant effect on ENaC activity. By using various inhibitors to probe Gβ1γ2 effects on ENaC regulation, we found that Gβ1γ2-mediated ENaC inhibition involved activation of phospholipase C-β and its enzymatic products that induce protein kinase C and ERK1/2 signaling pathways.

scholarly journals Structural basis for the ethanol action on G-protein–activated inwardly rectifying potassium channel 1 revealed by NMR spectroscopy

2018 ◽  
Vol 115 (15) ◽  
pp. 3858-3863 ◽  
Author(s):  
Yuki Toyama ◽  
Hanaho Kano ◽  
Yoko Mase ◽  
Mariko Yokogawa ◽  
Masanori Osawa ◽  
...  
Keyword(s):  
Nervous System ◽  
Nmr Spectroscopy ◽  
Potassium Channel ◽  
G Protein ◽  
Structural Basis ◽  
Signaling Proteins ◽  
Inwardly Rectifying Potassium Channel ◽  
Wide Range ◽  
Inwardly Rectifying ◽  
Human Nervous System

Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein–activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.

scholarly journals G Protein-Gated Inwardly Rectifying Potassium Channel Subunit 3 Knock-Out Mice Show Enhanced Ethanol Reward

2016 ◽  
Vol 40 (4) ◽  
pp. 857-864 ◽  
Author(s):  
Megan E. Tipps ◽  
Jonathan D. Raybuck ◽  
Laura B. Kozell ◽  
K. Matthew Lattal ◽  
Kari J. Buck
Keyword(s):  
Potassium Channel ◽  
G Protein ◽  
Knock Out ◽  
Inwardly Rectifying Potassium Channel ◽  
Knock Out Mice ◽  
Inwardly Rectifying
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