Functional Coupling Between Voltage-Dependent Sodium Channels and Activation of the Ca2+ Signaling That Mediates Endothelial Cell Migration

Angiogenesis depends on Ca²⁺-mediated endothelial cell migration. The increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is coordinated by caveolae and the Cx43 hemichannel opening. However, the functional coupling of voltage-dependent Na⁺ channels (Na_v) with Na⁺-Ca²⁺ exchanger reverse mode (NCXrm) activation may contribute to the response, which was evaluated using the wound-healing assay in primary cultures of rat mesenteric endothelial cells. Changes in [Ca²⁺]_i, the hemichannel opening and the association of Na_v channels with caveolin-1, a caveolae structural protein, were analyzed. Both endothelial cell migration and the associated Ca²⁺ signaling were inhibited by tetrodotoxin (TTX), a Na_v channel blocker, lamotrigine, a preferential Na_v1.2 inhibitor, or 4,9-anhydro-TTX, a specific Na_v1.6 blocker. A similar result was found by disrupting caveolae organization with methyl-β-cyclodextrin or blocking NCXrm with SEA0400. TTX and SEA0400 also prevented Cx43 hemichannel opening, and tubular-like structure formation depended on Na_v channels. An analysis using a proximity ligation assay showed that endothelial cell migration was paralleled by the progressive association of caveolin-1 with Na_v1.2, but not Na_v1.6, channels. These results suggest that the functional coupling of Na_v1.2 and Na_v1.6 channels with the activation of NCXrm and Cx43 hemichannels mediates the Ca²⁺ signaling associated with endothelial cell migration and angiogenesis, which provides new targets to modulate angiogenesis in physiological or pathological conditions.