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

dThis study was designed to elucidate the role of the subplasmalernmal 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,urn 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+](j); using fura-2) using a deconvolution imaging device. A voltage-dependent, large conductance Ca2+-dependent K+ channel (BKCa; single channel conductance (Y), 250 pS) was found. At membrane potentials of +40 and -40 mV, the EC50 for Ca2+ was 2.7 and 49.7 mum, respectively. In the vicinity of the sER, the BKCa channel activity induced by 10 muM 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 muM), BKCa 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 BKCa channel activity, ER Ca2+ depletion (Cam4-ER) and elevation of [Ca2+](i) in response to 10 and 100 muM 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.