Subplasmalemmal endoplasmic reticulum controls K(Ca) channel activity upon stimulation with a moderate histamine concentration in a human umbilical vein endothelial cell line.
This study was designed to elucidate the role of the subplasmalemmal endoplasmic reticulum (sER) in autacoid-induced stimulation of Ca(2+)-dependent K(+) channels in the umbilical vein endothelial cell-derived cell line EA.hy926. Cells were transfected with the Ca(2+) probe cameleon targeted to the ER for visualization of the ER network. A patch pipette was then placed close to or far (> 5 microm away) from the sER, single channel recordings (patch clamp technique) were monitored simultaneously with measurements of either ER Ca(2+) concentration (using the Ca(2+) probe Cam4-ER) or cytosolic free Ca(2+) concentration ([Ca(2+)](i); using fura-2) using a deconvolution imaging device. A voltage-dependent, large conductance Ca(2+)-dependent K(+) channel (BK(Ca); single channel conductance (gamma), 250 pS) was found. At membrane potentials of +40 and -40 mV, the EC(50) for Ca(2+) was 2.7 and 49.7 microM, respectively. In the vicinity of the sER, the BK(Ca) channel activity induced by 10 microM histamine was 32 times higher (open probability (P(o)) = 0.083 +/- 0.026) than in areas away from the sER (P(o) = 0.0026 +/- 0.002). However, at supramaximal histamine stimulation (100 microM), BK(Ca) channel activation was similar in patches in the vicinity of or away from the sER (P(o) = 0.18 +/- 0.09 and 0.25 +/- 0.07, respectively). In contrast to BK(Ca) channel activity, ER Ca(2+) depletion (Cam4-ER) and elevation of [Ca(2+)](i) in response to 10 and 100 microM histamine were not influenced by the pipette position. We conclude that in endothelial cells, the activation of BK(Ca) 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 Ca(2+) control unit (SCCU) and add the local activation of Ca(2+)-activated K(+)-channels to the function of the SCCU.
Frieden
Maud
M
Department of Medical Biochemistry and Medical Molecular Biology, Karl-Franzens University of Graz, Harrachgasse 21/III, A-8010 Graz, Austria.
Malli
Roland
R
Samardzija
Mariana
M
Demaurex
Nicolas
N
Graier
Wolfgang F
WF
D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D015335:Molecular Probes; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D015335:Molecular Probes; D024681:Potassium Channels, Calcium-Activated; D006632:Histamine; D011188:Potassium; D002118:Calcium