Na +/Ca2+ exchangers and Orai channels jointly refill endoplasmic reticulum (ER) Ca2+ via ER nanojunctions in vascular endothelial cells

Abstract

We investigated the role of Na⁺/ Ca²⁺ exchange (NCX) in the refilling of endoplasmic reticulum (ER) Ca²⁺ in vascular endothelial cells under various conditions of cell stimulation and plasma membrane (PM) polarization. Better understanding of the mechanisms behind basic ER Ca²⁺ content regulation is important, since current hypotheses on the possible ultimate causes of ER stress point to deterioration of the Ca²⁺ transport mechanism to/from ER itself. We measured [Ca²⁺]_i temporal changes by Fura-2 fluorescence under experimental protocols that inhibit a host of transporters (NCX, Orai, non-selective transient receptor potential canonical (TRPC) channels, sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA), Na⁺/ K⁺ ATPase (NKA)) involved in the Ca²⁺ communication between the extracellular space and the ER. Following histamine-stimulated ER Ca²⁺ release, blockade of NCX Ca²⁺-influx mode (by 10 μM KB-R7943) diminished the ER refilling capacity by about 40%, while in Orai1 dominant negative-transfected cells NCX blockade attenuated ER refilling by about 60%. Conversely, inhibiting the ouabain sensitive NKA (10 nM ouabain), which may be localized in PM-ER junctions, increased the ER Ca²⁺ releasable fraction by about 20%, thereby supporting the hypothesis that this process of privileged ER refilling is junction-mediated. Junctions were observed in the cell ultrastructure and their main parameters of membrane separation and linear extension were (9.6 ± 3.8) nm and (128 ± 63) nm, respectively. Our findings point to a process of privileged refilling of the ER, in which NCX and store-operated Ca²⁺ entry via the stromal interaction molecule (STIM)-Orai system are the sole protagonists. These results shed light on the molecular machinery involved in the function of a previously hypothesized subplasmalemmal Ca²⁺ control unit during ER refilling with extracellular Ca²⁺.