A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5)

2019 ◽  
Author(s):  
Boris Shalomov ◽  
Reem Handklo-Jamal ◽  
Haritha P. Reddy ◽  
Neta Theodor ◽  
Amal K. Bera ◽  
...  
Keyword(s):  
G Protein ◽  
Adrenal Cortex ◽  
Primary Aldosteronism ◽  
Loss Of Function ◽  
Open Probability ◽  
Pore Region ◽  
Gain Of Function ◽  
Whole Cell ◽  
Cytosolic Domains ◽  
Inwardly Rectifying

AbstractG-protein gated, inwardly rectifying potassium channels (GIRK) mediate inhibitory transmission in brain, heart, and adrenal cortex. GIRK4 (KCNJ5) subunits are abundant in the heart and adrenal cortex. Multiple mutations of KCNJ5 cause primary aldosteronism (PA). According to a leading concept, mutations in the pore region of GIRK4 cause loss of K+ selectivity; the ensuing Na+ influx depolarizes zona glomerulosa cells and activates voltage gated Ca2+ channels, inducing hypersecretion of aldosterone. The concept of selectivity loss has been extended to mutations in cytosolic domains of GIRK4 channels, remote from the pore region. We expressed GIRK4R52H, GIRK4E246K, and GIRK4G247R mutants in Xenopus oocytes and human adrenocortical carcinoma cell line (HAC15). Whole-cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K (but not GIRK1/4G247R) channels were greatly reduced compared to GIRK1/4WT. Nevertheless, all heterotetrameric mutants retained full K+ selectivity and inward rectification. When expressed as homotetramers, only GIRK4WT, but none of the mutants, produced whole-cell currents. Confocal imaging, single channel and Förster Resonance Energy Transfer (FRET) analyses showed: 1) reduction of membrane abundance of all mutated channels, especially as homotetramers, 2) impaired interaction with Gβγ subunits, and 3) reduced open probability of GIRK1/4R52H. VU0529331, a GIRK4 opener, activated homotetrameric GIRK4G247R channels, but not GIRK4R52H and GIRK4E246K. Our results suggest impaired gating (GIRK4R52H) and expression in plasma membrane (all mutants). We suggest that, contrary to the previously proposed mechanism, R52H and E246K mutants are loss-of-function rather than gain-of-function/selectivity-loss mutants. Hence, GIRK4 openers may be a potential course of treatment for patients with cytosolic N- and C-terminal mutations.Significance StatementMutations in KCNJ5 gene, which encodes for the GIRK4 subunit of G-protein inwardly rectifying K+ channels, are the main cause of primary aldosteronism, a major contributor to secondary hypertension. We report that three mutations in the cytosolic domain of GIRK4 cause loss-of-function, contrary to the prevailing concept that these mutations cause loss of selectivity and subsequent depolarization, i.e. essentially gain-of-function. Our findings correct the existing misconception regarding the biophysical mechanism that impairs the channel function, and may provide indications for future personalized treatment of the disease.

scholarly journals Label-Free Whole Cell Biosensing for High-Throughput Discovery of Activators and Inhibitors Targeting G Protein-Activated Inwardly Rectifying Potassium Channels

ACS Omega ◽  
2018 ◽  
Vol 3 (11) ◽  
pp. 14814-14823 ◽  
Author(s):  
Katrin M. Krebs ◽  
Eva M. Pfeil ◽  
Katharina Simon ◽  
Manuel Grundmann ◽  
Felix Häberlein ◽  
...  
Keyword(s):  
Potassium Channels ◽  
G Protein ◽  
High Throughput ◽  
Label Free ◽  
Whole Cell ◽  
Inwardly Rectifying Potassium Channels ◽  
Inwardly Rectifying

scholarly journals Disorders of the calcium-sensing receptor and partner proteins: insights into the molecular basis of calcium homeostasis

2016 ◽  
Vol 57 (3) ◽  
pp. R127-R142 ◽  
Author(s):  
Fadil M Hannan ◽  
Valerie N Babinsky ◽  
Rajesh V Thakker
Keyword(s):  
G Protein ◽  
Hormone Secretion ◽  
Urinary Calcium ◽  
Calcium Excretion ◽  
Loss Of Function ◽  
Mineral Density ◽  
Gain Of Function ◽  
Type 3

The extracellular calcium (Ca2+o)-sensing receptor (CaSR) is a family C G protein-coupled receptor, which detects alterations in Ca2+o concentrations and modulates parathyroid hormone secretion and urinary calcium excretion. The central role of the CaSR in Ca2+o homeostasis has been highlighted by the identification of mutations affecting the CASR gene on chromosome 3q21.1. Loss-of-function CASR mutations cause familial hypocalciuric hypercalcaemia (FHH), whereas gain-of-function mutations lead to autosomal dominant hypocalcaemia (ADH). However, CASR mutations are only detected in ≤70% of FHH and ADH cases, referred to as FHH type 1 and ADH type 1, respectively, and studies in other FHH and ADH kindreds have revealed these disorders to be genetically heterogeneous. Thus, loss- and gain-of-function mutations of the GNA11 gene on chromosome 19p13.3, which encodes the G-protein α-11 (Gα11) subunit, lead to FHH type 2 and ADH type 2, respectively; whilst loss-of-function mutations of AP2S1 on chromosome 19q13.3, which encodes the adaptor-related protein complex 2 sigma (AP2σ) subunit, cause FHH type 3. These studies have demonstrated Gα11 to be a key mediator of downstream CaSR signal transduction, and also revealed a role for AP2σ, which is involved in clathrin-mediated endocytosis, in CaSR signalling and trafficking. Moreover, FHH type 3 has been demonstrated to represent a more severe FHH variant that may lead to symptomatic hypercalcaemia, low bone mineral density and cognitive dysfunction. In addition, calcimimetic and calcilytic drugs, which are positive and negative CaSR allosteric modulators, respectively, have been shown to be of potential benefit for these FHH and ADH disorders.

Human Adrenal Glomerulosa Cells Express K2P and GIRK Potassium Channels that are inhibited by AngII and ACTH

2021 ◽  
Author(s):  
John J. Enyeart ◽  
Judith A. Enyeart
Keyword(s):  
G Protein ◽  
Zona Glomerulosa ◽  
Time Dependent ◽  
Aldosterone Secretion ◽  
Girk Channels ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Girk Channel ◽  
Inwardly Rectifying ◽  
G Protein Coupled

In whole-cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two pore (K2P) leak-type and a G-protein-coupled, inwardly-rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly-rectifying current was activated by arachidonic acid and inhibited by angiotensin II (AngII), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by AngII, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by AngII or ACTH, and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by AngII and ACTH are likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

scholarly journals A New Multisystem Disorder Caused by the Gαs Mutation p.F376V

2018 ◽  
Vol 104 (4) ◽  
pp. 1079-1089 ◽  
Author(s):  
Heike Biebermann ◽  
Gunnar Kleinau ◽  
Dirk Schnabel ◽  
Detlef Bockenhauer ◽  
Louise C Wilson ◽  
...  
Keyword(s):  
G Protein ◽  
De Novo ◽  
Clinical Findings ◽  
New Combination ◽  
Loss Of Function ◽  
Multisystem Disorder ◽  
Gain Of Function ◽  
Α Subunit ◽  
Serum Pth

Abstract Context The α subunit of the stimulatory G protein (Gαs) links numerous receptors to adenylyl cyclase. Gαs, encoded by GNAS, is expressed predominantly from the maternal allele in certain tissues. Thus, maternal heterozygous loss-of-function mutations cause hormonal resistance, as in pseudohypoparathyroidism type Ia, whereas somatic gain-of-function mutations cause hormone-independent endocrine stimulation, as in McCune-Albright syndrome. Objective We report two unrelated boys presenting with a new combination of clinical findings that suggest both gain and loss of Gαs function. Design and Setting Clinical features were studied and sequencing of GNAS was performed. Signaling capacities of wild-type and mutant Gαs were determined in the presence of different G protein–coupled receptors (GPCRs) under basal and agonist-stimulated conditions. Results Both unrelated patients presented with unexplained hyponatremia in infancy, followed by severe early onset gonadotrophin-independent precocious puberty and skeletal abnormalities. An identical heterozygous de novo variant (c.1136T>G; p.F376V) was found on the maternal GNAS allele in both patients; this resulted in a clinical phenotype that differed from known Gαs-related diseases and suggested gain of function at the vasopressin 2 receptor (V2R) and lutropin/choriogonadotropin receptor (LHCGR), yet increased serum PTH concentrations indicative of impaired proximal tubular PTH1 receptor (PTH1R) function. In vitro studies demonstrated that Gαs-F376V enhanced ligand-independent signaling at the PTH1R, LHCGR, and V2R and, at the same time, blunted ligand-dependent responses. Structural homology modeling suggested mutation-induced modifications at the C-terminal α5 helix of Gαs that are relevant for interaction with GPCRs and signal transduction. Conclusions The Gαs p.F376V mutation causes a previously unrecognized multisystem disorder.

scholarly journals Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (7) ◽  
pp. 3635-3643 ◽  
Author(s):  
H G Dohlman ◽  
D Apaniesk ◽  
Y Chen ◽  
J Song ◽  
D Nusskern
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Protein ◽  
Growth Arrest ◽  
Direct Interaction ◽  
Alpha Subunit ◽  
Protein Signaling ◽  
Loss Of Function ◽  
Gain Of Function ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

Genetic analysis of cell-cell signaling in Saccharomyces cerevisiae has led to the identification of a novel factor, known as Sst2p, that promotes recovery after pheromone-induced growth arrest (R. K. Chan and C. A. Otte, Mol. Cell. Biol. 2:11-20, 1982). Loss-of-function mutations lead to increased pheromone sensitivity, but this phenotype is partially suppressed by overexpression of the G protein alpha subunit gene (GPA1). Suppression is allele specific, however, suggesting that there is direct interaction between the two gene products. To test this model directly, we isolated and characterized several dominant gain-of-function mutants of SST2. These mutations block the normal pheromone response, including a loss of pheromone-stimulated gene transcription, cell cycle growth arrest, and G protein myristoylation. Although the SST2 mutations confer a pheromone-resistant phenotype, they do not prevent downstream activation by overexpression of G beta (STE4), a constitutively active G beta mutation (STE4Hpl), or a disruption of GPA1. None of the SST2 alleles affects the expression or stability of G alpha. These results point to the G protein alpha subunit as being the direct target of Sst2p action and underscore the importance of this novel desensitization factor in G-protein-mediated signaling.

Lamellipod extension and K+ current in osteoclasts are regulated by different types of G proteins

1994 ◽  
Vol 107 (2) ◽  
pp. 517-526 ◽  
Author(s):  
S.A. Arkett ◽  
S.J. Dixon ◽  
S.M. Sims
Keyword(s):  
Molecular Mass ◽  
G Protein ◽  
G Proteins ◽  
Actin Polymerization ◽  
Complete Suppression ◽  
Patch Clamp Technique ◽  
Membrane Area ◽  
Whole Cell ◽  
K Current ◽  
Inwardly Rectifying

Osteoclasts are the cells responsible for the resorption of bone and other mineralized tissues. GTP-binding proteins (G proteins) play important roles in regulating the activity of many cell types; however, there is limited knowledge of their functions in osteoclasts. We used the patch-clamp technique in the whole-cell configuration to introduce either hydrolysis-resistant guanosine triphosphate analogues or fluoroaluminate into single rat osteoclasts, and examined the effects of G protein activation on cell morphology and ionic conductances. Guanosine 5′-O-(3-thiotriphosphate) or 5′-guanylyl-imidodiphosphate, but not the control compounds adenosine 5′-O-(3-thiotriphosphate) or guanosine 5′-O-(2-thiodiphosphate), induced: (1) prompt spreading due to extension of lamellipodia; and (2) after a latency of several minutes, complete suppression of the inwardly rectifying K+ current. Pertussis toxin did not alter either spreading or suppression of K+ current induced by guanosine 5′-O-(3-thiotriphosphate). Cytochalasin D, but not colchicine, prevented guanosine 5′-O-(3-thiotriphosphate)-induced spreading, consistent with actin polymerization underlying lamellipod extension. Whole-cell capacitance did not change during guanosine 5′-O-(3-thiotriphosphate)-induced spreading, which is consistent with a lack of change in total plasma membrane area. Fluoroaluminate did not induce spreading, but it did suppress the K+ current. The differential effects of fluoroaluminate and guanosine 5′-O-(3-thiotriphosphate) suggest that lamellipod extension is regulated by a small molecular mass, monomeric G protein, whereas the inwardly rectifying K+ current is regulated by a large molecular mass, heterotrimeric G protein. Thus, osteoclast motility and ion transport are regulated by separate G protein-coupled pathways.

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.

G protein modulates thyroid hormone-induced Na+ channel activation in ventricular myocytes

2002 ◽  
Vol 283 (5) ◽  
pp. H2119-H2129 ◽  
Author(s):  
Luyi Sen ◽  
Yoshihide Sakaguchi ◽  
Guanggen Cui
Keyword(s):  
G Protein ◽  
Rate Constant ◽  
Pertussis Toxin ◽  
Ventricular Myocytes ◽  
Na Channel ◽  
Open Probability ◽  
Inactivation Curve ◽  
Pipette Solution ◽  
Whole Cell ◽  
State Rate

To evaluate the effects of liothyronine (3,5,3′-triiodo-l-thyronine, T3) on Na+ channel current ( I Na) properties, I Na was recorded in adult guinea pig ventricular myocytes. T3 (1 nM) acutely increased whole cell I Na and shifted the steady-state I Na inactivation curve dose dependently. When the pipette solution contained 100 μM GTP or GTPγS, the effect of T3 on the whole cell I Na was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T3 increased the open probability of single I Na by reducing the null probability. In the inside-out patch, T3 effect was 10 times faster than that in whole cell and cell-attached patches while GTPγS was present and could be completely washed out. T3 alone slightly increased the channel open probability by increasing the closed state to open state rate constant ( k CO) and reducing the null probability. GTPγS exposure only increased the number of functional channels. T3 and GTPγS synergistically enhanced the channel open probability 5.8 ± 0.5-fold by increasing k CO, decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T3 acts on the cytosolic side of the membrane and acutely activates I Na. Pertussis toxin-sensitive G protein modulation greatly magnifies the T3 effects on the channel kinetics and null probability, thereby increasing the channel open probability.

scholarly journals The results of immunohistochemical study of antibodies to CYP11B2 in primary hyperaldosteronism

2021 ◽  
Vol 18 (3) ◽  
pp. 245-253
Author(s):  
N. Yu. Romanova ◽  
L. S. Selivanova ◽  
N. M. Platonova ◽  
D. G. Beltsevich ◽  
E. A. Troshina
Keyword(s):  
Adrenal Cortex ◽  
Primary Aldosteronism ◽  
Somatic Mutations ◽  
Ct Scanning ◽  
Adrenal Tumors ◽  
Adrenocortical Adenoma ◽  
Loss Of Function ◽  
Functional Heterogeneity ◽  
Driver Genes ◽  
Tumor Area

Background: Aldosterone-producing adrenocortical adenoma (APA) is responsible for the majority of cases clinically diagnosed as primary aldosteronism. Aldosterone synthase (CYP11B2) is one of the enzymes that play essential roles in aldosterone synthesis and is involved in the pathogenesis of primary aldosteronism. Recent studies have demonstrated that various factors influence the expression and function of CYP11B2 in APA. In particular, somatic mutations, such as gain-of-function and loss-of-function mutations have been identified in several genes, each of which encodes a pivotal protein that affects the calcium signaling pathway, the expression of CYP11B2, and aldosterone production. On the other hand, CYP11B2 also catalyzes the conversion of cortisol to 18-hydroxycortisol and subsequently converts 18-hydroxycortisol to 18-oxocortisol. The article also discusses the clinical significance of 18-oxocortisol, an important biomarker for the diagnosis of primary aldosteronism. Somatic mutations in aldosterone-driver genes are strongly associated with CYP11B2 expression and have been only detected in the CYP11B2-positive tumor area, that indicating heterogeneous expression of CYP11B2 in tumor.Aim: to assess the functional heterogeneity of adrenal tumors in primary aldosteronismMaterials and methods: Retrospective evaluation adrenal tumors from patients with primary aldosteronism (n=20). According to CT unilateral macrohyperplasia was detected in 19 patients (95% of total), all of them were confirmed to have unilateral hyperproduction of aldosterone according to AVS. Selected tumors werestained with anty-CYP11B2 antibody. We evaluated the CYP11B2 expression in the adenoma and in the adjacent adrenal cortex.Results: Immunohistochemistry studies of the resected adrenals from 20 patients with PA operated due to unilateral production of aldosterone using CYP11B2 staining showed that 10 of those with an adenoma on CT scanning showed CYP11B2 staining in the adenoma. Furthermore, 5 cases of an unilateral adenoma, showed CYP11B2 staining in the adjacent adrenal cortex and an absence of staining for CYP11B2 in the adenoma. 5 cases showed CYP11B2 expression is heterogeneously immunolocalized throughout the tumor area.Conclusion: Thus, the functional heterogeneity of adrenal tumors in primary aldosteronism has been proven. It is necessary to compare the data of immunohistochemical studies on the expression of CYP11B2 with the indicators of the level of 18-hydroxycortisol, 18-oxocortisol.

A mutation in the β-subunit of ENaC identified in a patient with cystic fibrosis-like symptoms has a gain-of-function effect

2013 ◽  
Vol 304 (1) ◽  
pp. L43-L55 ◽  
Author(s):  
Robert Rauh ◽  
Daniel Soell ◽  
Silke Haerteis ◽  
Alexei Diakov ◽  
Viatcheslav Nesterov ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Channel Modeling ◽  
Hek293 Cells ◽  
Β Subunit ◽  
Open Probability ◽  
Gain Of Function ◽  
Proteolytic Activation ◽  
Whole Cell ◽  
Cell Current ◽  
Whole Cell Current

In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the β-subunit of ENaC (βV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αβγENaC or mutant αβV348MγENaC in Xenopus laevis oocytes. The βV348M mutation stimulated amiloride-sensitive whole-cell current (Δ Iami) by ∼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability ( Po). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (∼3.0-fold vs. ∼4.2-fold), which is consistent with the increased baseline Po of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by ∼2.6-fold) than wt ENaC currents (by ∼3.5-fold). The gain-of-function effect of the βV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different βV348 mutants in oocytes suggests that the βV348M mutation increases channel Po by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the βV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.

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