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

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+. Electronic supplementary material The online version of this article (doi:10.1007/s00424-017-1989-8) contains supplementary material, which is available to authorized users.

S2 ER Ca 2+ releasable fraction in absence of normal Ca 2+ phase (nCa 2+ ) The data reported in the solid red circles and red bar in figure S2A and B are the same as in figure   2C and D (also in red) in the main article. They represent the Fura-2 signal during a series of experiments in absence of the nCa 2+ phase of the protocol (see top part of figure S2A).
In the experiments performed to obtain the data in empty green circles in figure S2A and B, we added the reversible SERCA blocker 2,5-Di-tert-butyl-1,4-benzoquinone (BHQ) during the first histamine stimulation period to prevent the Ca 2+ released from the ER from being re-uptaken while still present in free form in the cytosol.
These results show that the majority of the ER releasable Ca 2+ remaining after a 3-min histamine stimulation is due to internal Ca 2+ "recycling" during the stimulation itself and confirm that the ER releasable Ca 2+ fraction is virtually entirely refilled from the extra-cellular space. Bar chart from traces in A. *** indicates p<0.001.

S2
S3 Smearing of the PM in the electron micrographs of PM-

ER nanojunctions
The identification and characterization of PM-ER junctions in cultured cells like the EA.hy926 endothelial cells employed in our experiments can be hampered by the smearing of the PM due to the microtome sectioning phase of the sample preparation.
In figure S3 we report samples of micrographs from the set we collected showing instances of PM-ER nanojunctions, in which the PM is well defined (green arrow) thereby making measurement of the junctional gap and extension possible. In cases when the PM results smeared (red arrows) this is not possible and therefore the number of junctions we report in the histograms of the main article is likely an underestimate of the actual number.
The smearing effect is probably due to the fact that cultured cells lie flat on the sample substrate and present a cross-section that is bulbous at the location of the nucleus and gets progressively thinner toward the cell periphery. In these conditions, when sectioning the samples in a direction parallel to the cell substrate it becomes less likely that the knife cuts the PM as cleanly as other intracellular membranes lying in the thicker part of the cells. For this reason, we also opted to re-orient the samples so that they could be sectioned in a direction perpendicular to the substrate plane.

S3
S4 TPRC channels appear not to contribute to ER Ca 2+ refilling Transient receptor potential canonical (TRPC) channels appear to contribute only to the bulk cytosolic Ca 2+ in EA.hy926 endothelial cells, but not measurably to the ER Ca 2+ content. In normally polarized cells (empty black circles in figure S4), Ca 2+ entry via TRPC is virtually unimpaired (except perhaps for some interference by the unspecificity of KB-R7943). In this situation, while inhibiting NCX Ca 2+ entry mode (with 10 µM KB-R7943), our data show that the Ca 2+ amplitude transient ratio between the cytosolic signal and the ER Ca 2+ releasable fraction is significantly higher than in depolarized cells (∆R 2nd /∆R 3rd in figure S4), while the ER releasable Ca 2+ fraction is not appreciably different (∆R 3rd /∆R 1st in figure S4).