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 (Nav) 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 Nav 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 Nav channel blocker, lamotrigine, a preferential Nav1.2 inhibitor, or 4,9-anhydro-TTX, a specific Nav1.6 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 Nav channels. An analysis using a proximity ligation assay showed that endothelial cell migration was paralleled by the progressive association of caveolin-1 with Nav1.2, but not Nav1.6, channels. These results suggest that the functional coupling of Nav1.2 and Nav1.6 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.