RT Journal Article
SR Electronic
T1 Na<sup>+</sup>/Ca<sup>2+</sup> exchangers and Orai channels jointly refill endoplasmic reticulum (ER) Ca<sup>2+</sup> via ER nanojunctions in vascular endothelial cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 084285
DO 10.1101/084285
A1 Cristiana M. L. Di Giuro
A1 Niroj Shrestha
A1 Roland Malli
A1 Klaus Groschner
A1 Cornelis van Breemen
A1 Nicola Fameli
YR 2016
UL http://biorxiv.org/content/early/2016/10/28/084285.abstract
AB 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 high-ouabain-affinity 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 SOCE 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+.