Subplasmalemmal endoplasmic reticulum controls K_Ca channel activity upon stimulation with a moderate histamine concentration in a human umbilical vein endothelial cell line

Abstract

This study was designed to elucidate the role of the subplasmalemmal endoplasmic reticulum (sER) in autacoid‐induced stimulation of Ca2+‐dependent K+ channels in the umbilical vein endothelial cell‐derived cell line EA.hy926. Cells were transfected with the Ca2+ probe cameleon targeted to the ER for visualization of the ER network. A patch pipette was then placed close to or far (> 5 μm away) from the sER, single channel recordings (patch clamp technique) were monitored simultaneously with measurements of either ER Ca2+ concentration (using the Ca2+ probe Cam4‐ER) or cytosolic free Ca2+ concentration ([Ca2+]i; using fura‐2) using a deconvolution imaging device. A voltage‐dependent, large conductance Ca2+‐dependent K+ channel (BKCa; single channel conductance (γ), 250 pS) was found. At membrane potentials of +40 and −40 mV, the EC50 for Ca2+ was 2.7 and 49.7 μm, respectively. In the vicinity of the sER, the BKCa channel activity induced by 10 μm histamine was 32 times higher (open probability (Po) = 0.083 ± 0.026) than in areas away from the sER (Po= 0.0026 ± 0.002). However, at supramaximal histamine stimulation (100 μm), BKCa channel activation was similar in patches in the vicinity of or away from the sER (Po= 0.18 ± 0.09 and 0.25 ± 0.07, respectively). In contrast to BKCa channel activity, ER Ca2+ depletion (Cam4‐ER) and elevation of [Ca2+]i in response to 10 and 100 μm histamine were not influenced by the pipette position. We conclude that in endothelial cells, the activation of BKCa channels in response to moderate histamine concentration essentially depends on the proximity of the sER domains to the mouth of this K+ channel. These findings further support our concept of the subplasmalemmal Ca2+ control unit (SCCU) and add the local activation of Ca2+‐activated K+‐channels to the function of the SCCU.

Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum

“…In addition, to avoid contamination by constitutively expressed LPL, cells lacking endogenous LPL [34,35] were infected. Accordingly, because of our extensive experience on the Ca 2+ homoeostasis in the human umbilical vein derived endothelial cell line EA.hy926 [2,3,15,29,36], these cells were chosen as a suitable model system.…”

“…Intraluminal free Ca 2+ concentration was monitored as described previously [3,29]. In brief, at a confluence of approx.…”

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

“…Imaging Device-Our setup for monitoring cytosolic and organelle Ca 2ϩ signaling was described previously (24). All of the Ca 2ϩ measurements were performed at room temperature on a Nikon inverted microscope (Eclipse 300 TE, Nikon, Vienna, Austria) equipped with an epifluorescence system (150 W XBO, Optiquip, Highland Mills, NY) using the Plan Fluor ϫ40 oil immersion objective from Nikon.…”

“…Recently, we demonstrated that superficial mitochondria are able to efficiently buffer the subplasmalemmal Ca 2ϩ concentration ([Ca 2ϩ ] pm ) beneath the plasma membrane (22) despite a strong agonist-evoked cytosolic Ca 2ϩ elevation and the generation of high Ca 2ϩ levels between the plasma membrane and superficial ER domains (i.e. subplasmalemmal Ca 2ϩ control unit, SCCU) (22)(23)(24). Hence, mitochondrial Ca 2ϩ uptake goes along with vectorial Ca 2ϩ release via mitochondrial Na ϩ /Ca 2ϩ exchanger (NCX mito ) toward the ER and the cytosol that becomes visible by ER Ca 2ϩ refilling and the elevation in [Ca 2ϩ ] cyto , respectively (19).…”

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells

“…Although the ER extends throughout the cell, the membraneenclosed cisternae just underneath the plasma membrane termed sPL-sER or when in close apposition to the plasma membrane, also called "junctional ER" (jER) has been implicated in specific functions related to Ca 2ϩ fluxes (Graier et al, 1998;Frieden et al, 2002;Malli et al, 2003). Since an almost twofold increase in peripheral mitochondrial volume density was observed, we investigated, whether additionally changes in sPL-sER have occurred in PC somata of mice deficient for PV, CB or both.…”