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Cell Depolarization (cell + depolarization)
Selected AbstractsMyosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretionJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Sergio D. Rosé Abstract The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine ,-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks. [source] Membrane potential and endocytic activity control disintegration of cell,cell adhesion and cell fusion in vinculin-injected MDBK cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Riitta Palovuori Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961,974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell,cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin,membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin,membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local and transient pH gradients play a role. © 2004 Wiley-Liss, Inc. [source] Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretionJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Sergio D. Rosé Abstract The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine ,-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks. [source] The K+,Cl, cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampusTHE JOURNAL OF PHYSIOLOGY, Issue 9 2010Tero Viitanen GABAergic excitatory [K+]o transients can be readily evoked in the mature rat hippocampus by intense activation of GABAA receptors (GABAARs). Here we show that these [K+]o responses induced by high-frequency stimulation or GABAA agonist application are generated by the neuronal K+,Cl, cotransporter KCC2 and that the transporter-mediated KCl extrusion is critically dependent on the bicarbonate-driven accumulation of Cl, in pyramidal neurons. The mechanism underlying GABAergic [K+]o transients was studied in CA1 stratum pyramidale using intracellular sharp microelectrodes and extracellular ion-sensitive microelectrodes. The evoked [K+]o transients, as well as the associated afterdischarges, were strongly suppressed by 0.5,1 mm furosemide, a KCl cotransport inhibitor. Importantly, the GABAAR-mediated intrapyramidal accumulation of Cl,, as measured by monitoring the reversal potential of fused IPSPs, was unaffected by the drug. It was further confirmed that the reduction in the [K+]o transients was not due to effects of furosemide on the Na+ -dependent K+ -Cl, cotransporter NKCC1 or on intraneuronal carbonic anhydrase activity. Blocking potassium channels by Ba2+ enhanced [K+]o transients whereas pyramidal cell depolarizations were attenuated in further agreement with a lack of contribution by channel-mediated K+ efflux. The key role of the GABAAR channel-mediated anion fluxes in the generation of the [K+]o transients was examined in experiments where bicarbonate was replaced with formate. This anion substitution had no significant effect on the rate of Cl, accumulation, [K+]o response or afterdischarges. Our findings reveal a novel excitatory mode of action of KCC2 that can have substantial implications for the role of GABAergic transmission during ictal epileptiform activity. [source] | ||||||||||