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⁺.

Keywords: Calcium signals; EA.hy926; ER junctions; Endothelium; NCX; Orai1.

Fig. 1

Top left: schematic partial cross-section of a vessel with location of EC (image attribution: DRosenbach at English Wikipedia); top right: diagram of one EC showing location of PM-ER junctions; bottom: essential depiction of the current understanding of the Ca²⁺ transport machinery, ionic fluxes and some of the mechanisms responsible for ER Ca²⁺ refilling in EC. (N = nucleus; ROC = receptor operated channels; SOC = store operated channels; see text for explanations of the other acronyms)

Fig. 2

a Schematics of the possible ion fluxes at PM-ER junctions between the extra-cellular space and ER under 70-mM- [K⁺]_o depolarizing condition. b Main protocol results under normal membrane polarization conditions: [Ca²⁺]_o and histamine stimulation timeline (top), recorded Fura-2 signal (bottom). c Effect of depolarization on Ca²⁺ entry and ER refilling (solid blue circles); effect of n Ca²⁺ phase omission on the Ca²⁺ signal (open red circles). d Bar chart representing the ratio between 3^rd and 1^st Ca²⁺ transient amplitudes from traces in b, c. *** indicates p < 0.001

Fig. 3

a Schematics of the possible ion fluxes at PM-ER junction locations during the NCX Ca²⁺ influx mode inhibition experiments. b Effect of 10 μM KB-R7943 on ER refilling (open yellow circles); recordings from our standard protocol (solid blue circles) reported as reference. c Bar chart from traces in b; left, ratio between 3^rd and 1^st Ca²⁺ transient amplitudes; right, ratio between 2^nd and 3^rd amplitudes. d Bar chart from traces in B, comparing the Ca²⁺ re-entry rates into the cytoplasm at the start of the n Ca²⁺ phase of the protocol. *** indicates p < 0.001

Fig. 4

a Schematics of the possible ion fluxes at PM-ER junctions during inhibition of ouabain sensitive NKAα _2/3 pumps. b Effect of 10 nM ouabain on ER refilling (open orange circles); recordings from our standard protocol (solid blue circles) reported as reference. c Bar chart from traces in b; left, ratio between 3^rd and 1^st Ca²⁺ transient amplitudes; right, ratio between 2^nd and 3^rd Ca²⁺ transient amplitudes. d Bar chart from traces in b, comparing the Ca²⁺ re-entry rates at the start of the n Ca²⁺ phase of the protocol. *** indicates p < 0.001

Fig. 5

a Schematics of the possible ion fluxes in PM-ER junctional regions during NCX inhibition in Orai1^dn-transfected cells. b Effect of 10 μM KB-R7943 on ER refilling in absence of SOCE contribution by the STIM-Orai1 system (open purple circles); recordings from our standard protocol (solid blue circles) reported as reference. c Bar chart representing the ratio between 3^rd and 1^st Ca²⁺ transient amplitudes from traces in b. *** indicates p < 0.001

Fig. 6

a Representative electron micrograph of a peripheral region of an EA.hy926 cultured EC; the right side panel is a higher resolution image of part of the same region showing a PM-ER junction near a caveola and several instances of junction-spanning electron opaque structures (white arrowheads; cyt = cytoplasm, cav = caveola). b, c Histograms showing the distributions of PM-ER widths and lateral extensions, respectively

Fig. 7

NCX reversal potential vs [Ca²⁺]_i, V _m under depolarization (70 mM K⁺) conditions as measured in [41] (blue line) and calculated by the Goldman’s equation (green)