scholarly journals Targeting of Intracellular TMEM16 Proteins to the Plasma Membrane and Activation by Purinergic Signaling

2020 ◽  
Vol 21 (11) ◽  
pp. 4065
Author(s):  
Rainer Schreiber ◽  
Jiraporn Ousingsawat ◽  
Karl Kunzelmann
Keyword(s):  
Plasma Membrane ◽  
Ion Channels ◽  
Purinergic Receptor ◽  
Purinergic Signaling ◽  
P2x7 Receptors ◽  
Membrane Blebbing ◽  
Functional Studies ◽  
Intracellular Compartments ◽  
Cl Channels ◽  
Stimulation Of

Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl− channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and TMEM16B form halide-selective ion channels, TMEM16F and probably TMEM16E operate as phospholipid scramblases and nonselective ion channels. Other TMEM16 paralogs are expressed mainly in intracellular compartments and are therefore difficult to study at the functional level. Here, we report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX (cysteine, two aliphatic amino acids plus methionin, serine, alanin, cystein or glutamin) motif. These paralogs produce Ca2+-dependent ion channels. Surprisingly, expression of the TMEM16 paralogs in the plasma membrane did not produce additional scramblase activity. In contrast, endogenous scrambling induced by stimulation of purinergic P2X7 receptors was attenuated, in parallel with reduced plasma membrane blebbing. This could suggest that intracellular TMEM16 paralogs operate differently when compared to plasma membrane-localized TMEM16F, and may even stabilize intracellular membranes. Alternatively, CAAX tagging, which leads to expression in non-raft compartments of the plasma membrane, may antagonize phosphatidylserine exposure by endogenous raft-located TMEM16F. CAAX-containing constructs may be useful to further investigate the molecular properties of intracellular TMEM16 proteins.

scholarly journals Insulin Stimulation of GLUT4 Exocytosis, but Not Its Inhibition of Endocytosis, Is Dependent on RabGAP AS160

2004 ◽  
Vol 15 (10) ◽  
pp. 4406-4415 ◽  
Author(s):  
Anja Zeigerer ◽  
Mary Kate McBrayer ◽  
Timothy E. McGraw
Keyword(s):  
Plasma Membrane ◽  
Blood Glucose ◽  
Insulin Signaling ◽  
Glucose Transporter ◽  
Whole Body ◽  
Insulin Stimulation ◽  
Molecular Events ◽  
Intracellular Compartments ◽  
Blood Glucose Homeostasis ◽  
Stimulation Of

Insulin maintains whole body blood glucose homeostasis, in part, by regulating the amount of the GLUT4 glucose transporter on the cell surface of fat and muscle cells. Insulin induces the redistribution of GLUT4 from intracellular compartments to the plasma membrane, by stimulating a large increase in exocytosis and a smaller inhibition of endocytosis. A considerable amount is known about the molecular events of insulin signaling and the complex itinerary of GLUT4 trafficking, but less is known about how insulin signaling is transmitted to GLUT4 trafficking. Here, we show that the AS160 RabGAP, a substrate of Akt, is required for insulin stimulation of GLUT4 exocytosis. A dominant-inhibitory mutant of AS160 blocks insulin stimulation of exocytosis at a step before the fusion of GLUT4-containing vesicles with the plasma membrane. This mutant, however, does not block insulin-induced inhibition of GLUT4 endocytosis. These data support a model in which insulin signaling to the exocytosis machinery (AS160 dependent) is distinct from its signaling to the internalization machinery (AS160 independent).

scholarly journals Reversible Ca Gradients between the Subplasmalemma and Cytosol Differentially Activate Ca-dependent Cl Currents

1999 ◽  
Vol 113 (2) ◽  
pp. 249-266 ◽  
Author(s):  
Khaled Machaca ◽  
H. Criss Hartzell
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Driving Force ◽  
Xenopus Oocytes ◽  
Biophysical Properties ◽  
Temporal Features ◽  
Cl Channels ◽  
Spatio Temporal ◽  
Stimulation Of

Xenopus oocytes express several different Ca-activated Cl currents that have different waveforms and biophysical properties. We compared the stimulation of Ca-activated Cl currents measured by two-microelectrode voltage clamp with the Ca transients measured in the same cell by confocal microscopy and Ca-sensitive fluorophores. The purpose was to determine how the amplitude and/or spatio-temporal features of the Ca signal might explain how these different Cl currents were activated by Ca. Because Ca release from stores was voltage independent, whereas Ca influx depended upon the electrochemical driving force, we were able to separately assess the contribution of Ca from these two sources. We were surprised to find that Ca signals measured with a cytosolic Ca-sensitive dye, dextran-conjugated Ca-green-1, correlated poorly with Cl currents. This suggested that Cl channels located at the plasma membrane and the Ca-sensitive dye located in the bulk cytosol were sensing different [Ca]. This was true despite Ca measurement in a confocal slice very close to the plasma membrane. In contrast, a membrane-targeted Ca-sensitive dye (Ca-green-C18) reported a Ca signal that correlated much more closely with the Cl currents. We hypothesize that very local, transient, reversible Ca gradients develop between the subplasmalemmal space and the bulk cytosol. [Ca] is higher near the plasma membrane when Ca is provided by Ca influx, whereas the gradient is reversed when Ca is released from stores, because Ca efflux across the plasma membrane is faster than diffusion of Ca from the bulk cytosol to the subplasmalemmal space. Because dissipation of the gradients is accelerated by inhibition of Ca sequestration into the endoplasmic reticulum with thapsigargin, we conclude that [Ca] in the bulk cytosol declines slowly partly due to futile recycling of Ca through the endoplasmic reticulum.

scholarly journals Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD

2019 ◽  
Vol 317 (6) ◽  
pp. F1649-F1655 ◽  
Author(s):  
Sergey N. Arkhipov ◽  
D’Anna L. Potter ◽  
Aron M. Geurts ◽  
Tengis S. Pavlov
Keyword(s):  
Purinergic Receptor ◽  
Kidney Diseases ◽  
Purinergic Signaling ◽  
Experimental Studies ◽  
Sodium Reabsorption ◽  
Channel Activity ◽  
P2x7 Receptors ◽  
Exon 2 ◽  
Pannexin 1 ◽  
Cyst Growth

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK. P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK. P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.

scholarly journals Pharmacological Approaches for the Modulation of the Potassium Channel KV4.x and KChIPs

2021 ◽  
Vol 22 (3) ◽  
pp. 1419
Author(s):  
Pilar Cercós ◽  
Diego A. Peraza ◽  
Angela de Benito-Bueno ◽  
Paula G. Socuéllamos ◽  
Abdoul Aziz-Nignan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ion Channels ◽  
Hippocampal Neurons ◽  
Outward Current ◽  
Underlying Mechanism ◽  
Transient Outward Current ◽  
Regulatory Subunits ◽  
Functional Studies ◽  
Accessory Subunits ◽  
Complex Knowledge

Ion channels are macromolecular complexes present in the plasma membrane and intracellular organelles of cells. Dysfunction of ion channels results in a group of disorders named channelopathies, which represent an extraordinary challenge for study and treatment. In this review, we will focus on voltage-gated potassium channels (KV), specifically on the KV4-family. The activation of these channels generates outward currents operating at subthreshold membrane potentials as recorded from myocardial cells (ITO, transient outward current) and from the somata of hippocampal neurons (ISA). In the heart, KV4 dysfunctions are related to Brugada syndrome, atrial fibrillation, hypertrophy, and heart failure. In hippocampus, KV4.x channelopathies are linked to schizophrenia, epilepsy, and Alzheimer’s disease. KV4.x channels need to assemble with other accessory subunits (β) to fully reproduce the ITO and ISA currents. β Subunits affect channel gating and/or the traffic to the plasma membrane, and their dysfunctions may influence channel pharmacology. Among KV4 regulatory subunits, this review aims to analyze the KV4/KChIPs interaction and the effect of small molecule KChIP ligands in the A-type currents generated by the modulation of the KV4/KChIP channel complex. Knowledge gained from structural and functional studies using activators or inhibitors of the potassium current mediated by KV4/KChIPs will better help understand the underlying mechanism involving KV4-mediated-channelopathies, establishing the foundations for drug discovery, and hence their treatments.

Non-contact long-range magnetic stimulation of mechanosensitive ion channels in freely moving animals

2021 ◽  
Author(s):  
Jung-uk Lee ◽  
Wookjin Shin ◽  
Yongjun Lim ◽  
Jungsil Kim ◽  
Woon Ryoung Kim ◽  
...  
Keyword(s):  
Ion Channels ◽  
Long Range ◽  
Magnetic Stimulation ◽  
Mechanosensitive Ion Channels ◽  
Freely Moving ◽  
Stimulation Of

Stimulation of Tumor-Specific Immunity Using Tumor Cell Plasma Membrane Antigen

Methods ◽  
1997 ◽  
Vol 12 (2) ◽  
pp. 155-164 ◽  
Author(s):  
Matthew F Mescher ◽  
Elena Savelieva
Keyword(s):  
Plasma Membrane ◽  
Tumor Cell ◽  
Cell Plasma Membrane ◽  
Specific Immunity ◽  
Cell Plasma ◽  
Stimulation Of

scholarly journals Redistribution of alpha-granules and their contents in thrombin-stimulated platelets.

1984 ◽  
Vol 98 (2) ◽  
pp. 748-760 ◽  
Author(s):  
P E Stenberg ◽  
M A Shuman ◽  
S P Levine ◽  
D F Bainton
Keyword(s):  
Plasma Membrane ◽  
Tannic Acid ◽  
Extracellular Space ◽  
Plasma Membranes ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Thin Sections ◽  
Immunocytochemical Techniques ◽  
Morphologic Changes ◽  
Stimulation Of

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.

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