Supplemental data and data sets for: Extracellular Na+ and Orai1 contribute to endothelial wound healing (Xu et al)

Endothelial migration is necessary for wound healing and angiogenesis. Na+ has been incriminated as an important cellular signal mediating migration, although the ion channels regulating Na+ permeation that contribute to migration remain poorly understood. We have shown that Orai1 suppresses both constitutive Na+ leak and Na+
entry through store-operated calcium entry channels. Here, we examine
the independent and combined contributions of extracellular Na+
and Orai1 to pulmonary artery endothelial cell (PAEC) wound healing. We
found that PAEC monolayers migrated into wound areas and achieved
confluence within 24 hours post-scratch. There was a fast healing phase
within 5 hours, followed by a slow-healing phase between 5 and 24 hours.
Na+ depletion reduced migration speed by at least 50%. Fewer
cells at the migration front formed prominent lamellipodia. Some cells
at the leading edge gradually detached from the monolayer. Reduction of
Orai1 decreased wound closure speed by 25% within 6 hours. This
Orai1-dependence was absent in Na+-depleted media and after 6
hours. In a non-scratch cell migration assay, the gap areas were
partially sealed within 24 hours. Migration speed was reduced by 75%
when extracellular Na+ was replaced by choline. Orai1 did not contribute to non-scratch cell migration, but extracellular Na+ did. Overall, extracellular Na+ and Orai1 together acutely regulate endothelial wound healing. Extracellular Na+ continuously contributes to wound healing between 6 and 24 hours. Extracellular Na+
but not Orai1 contributes to cell migration in the absence of injury.
Orai1 may function as an injury sensor to acutely stimulate fast wound
healing.