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 The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR

2016 ◽  
Vol 215 (4) ◽  
pp. 543-558 ◽  
Author(s):  
Sandra Scharaw ◽  
Murat Iskar ◽  
Alessandro Ori ◽  
Gaelle Boncompain ◽  
Vibor Laketa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Egf Receptor ◽  
Regulatory Mechanism ◽  
Transcriptional Regulator ◽  
Transport Efficiency ◽  
Early Endosomes ◽  
Epidermal Growth ◽  
Partial Degradation ◽  
Stimulation Of

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.

scholarly journals The sarcoplasmic–endoplasmic reticulum Ca2+ ATPase 2b regulates the Ca2+ transients elicited by P2Y2 activation in PC Cl3 thyroid cells

2006 ◽  
Vol 190 (3) ◽  
pp. 641-649 ◽  
Author(s):  
Luca Ulianich ◽  
Maria Giovanna Elia ◽  
Antonella Sonia Treglia ◽  
Antonella Muscella ◽  
Bruno Di Jeso ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Thyroid Cell ◽  
Myristate Acetate ◽  
Thyroid Cells ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Thyroid Cell Line ◽  
Rat Thyroid ◽  
Stimulation Of

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]i level and increased the peak of Ca2+ entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+ and Ba2+ uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+ exchanger was not implicated, since the rate of decrement to the basal [Ca2+]i level was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+ transients caused by P2Y2 stimulation.

scholarly journals Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle

1985 ◽  
Vol 231 (3) ◽  
pp. 737-742 ◽  
Author(s):  
J Verbist ◽  
F Wuytack ◽  
L Raeymaekers ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Transport Atpase ◽  
Calmodulin Binding ◽  
Dependent Atpase ◽  
Inhibitory Antibodies ◽  
Stimulation Of

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

scholarly journals Intracellular Ca2+Release Triggers Translocation of Membrane Marker FM1–43 from the Extracellular Leaflet of Plasma Membrane into Endoplasmic Reticulum in T Lymphocytes

2005 ◽  
Vol 280 (16) ◽  
pp. 16377-16382 ◽  
Author(s):  
Sepehr Dadsetan ◽  
Vyacheslav Shishkin ◽  
Alla F. Fomina
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
T Cell ◽  
T Cell Receptor ◽  
Membrane Trafficking ◽  
Cell Receptor ◽  
Multivesicular Bodies ◽  
Close Proximity ◽  
Intracellular Ca ◽  
Stimulation Of

Stimulation of T cell receptor in lymphocytes enhances Ca2+signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1–43 to explore membrane trafficking upon mobilization of intracellular Ca2+in Jurkat T cells. We established that liberation of intracellular Ca2+with T cell receptor agonist phytohemagglutinin P or with Ca2+-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1–43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1–43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1–43-containing ER were free of dye. Intralumenal FM1–43 accumulation was observed even when Ca2+signaling in the cytosol was abolished by the removal of extracellular Ca2+. Our findings strongly suggest that release of intracellular Ca2+may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca2+elevation.

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.

Combining Optogenetic Stimulation and Motor Training Improves Functional Recovery and Perilesional Cortical Activity

2021 ◽  
pp. 154596832110566
Author(s):  
Emilia Conti ◽  
Alessandro Scaglione ◽  
Giuseppe de Vito ◽  
Francesco Calugi ◽  
Maria Pasquini ◽  
...  
Keyword(s):  
Functional Recovery ◽  
Combined Therapy ◽  
Motor Training ◽  
Motor Representation ◽  
Rehabilitative Treatment ◽  
Optogenetic Stimulation ◽  
Temporal Features ◽  
Spatio Temporal ◽  
Motor Representations ◽  
Stimulation Of

Background. An ischemic stroke is followed by the remapping of motor representation and extensive changes in cortical excitability involving both hemispheres. Although stimulation of the ipsilesional motor cortex, especially when paired with motor training, facilitates plasticity and functional restoration, the remapping of motor representation of the single and combined treatments is largely unexplored. Objective. We investigated if spatio-temporal features of motor-related cortical activity and the new motor representations are related to the rehabilitative treatment or if they can be specifically associated to functional recovery. Methods. We designed a novel rehabilitative treatment that combines neuro-plasticizing intervention with motor training. In detail, optogenetic stimulation of peri-infarct excitatory neurons expressing Channelrhodopsin 2 was associated with daily motor training on a robotic device. The effectiveness of the combined therapy was compared with spontaneous recovery and with the single treatments (ie optogenetic stimulation or motor training). Results. We found that the extension and localization of the new motor representations are specific to the treatment, where most treatments promote segregation of the motor representation to the peri-infarct region. Interestingly, only the combined therapy promotes both the recovery of forelimb functionality and the rescue of spatio-temporal features of motor-related activity. Functional recovery results from a new excitatory/inhibitory balance between hemispheres as revealed by the augmented motor response flanked by the increased expression of parvalbumin positive neurons in the peri-infarct area. Conclusions. Our findings highlight that functional recovery and restoration of motor-related neuronal activity are not necessarily coupled during post-stroke recovery. Indeed the reestablishment of cortical activation features of calcium transient is distinctive of the most effective therapeutic approach, the combined therapy.

scholarly journals Calcium influx in mammalian eggs

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. R97-R105 ◽  
Author(s):  
Chunmin Wang ◽  
Zoltan Machaty
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Assisted Reproductive Technologies ◽  
Calcium Influx ◽  
Critical Role ◽  
Reproductive Technologies ◽  
Signaling Cascades ◽  
Mammalian Eggs ◽  
Stimulation Of

Calcium (Ca2+) signals are involved in the regulation of oocyte maturation and play a critical role during fertilization. In the egg, Ca2+is stored in the lumen of the endoplasmic reticulum and a signal is generated when the stored Ca2+is released through specialized channels in the membrane of the endoplasmic reticulum to elevate the free Ca2+concentration in the cytoplasm. Extracellular Ca2+is also important, indicated by the fact that the mobilization of luminal Ca2+is typically followed by Ca2+entry across the plasma membrane. The transmembrane Ca2+flux replenishes the endoplasmic reticulum, and thus, it is essential to sustain prolonged Ca2+signals. It also seems to be responsible for the stimulation of important signaling cascades required for complete egg activation. Characterization of the pathway that mediates Ca2+entry implies that its major components include STIM1, a protein that senses the filling status of the stores, and ORAI1, a channel protein located in the plasma membrane. Defining the mechanism and functions of Ca2+entry will not only lead to a better understanding of egg physiology but may also help improving the efficiency of a number of assisted reproductive technologies.

Triple Fixation For Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 90-91 ◽  
Author(s):  
M. A. Hayat
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Plasma Membrane ◽  
Myelin Sheath ◽  
Good Deal ◽  
Granular Structure ◽  
Oxidizing Agent ◽  
Fixation Technique ◽  
Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

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