Membrane potential (Vm) of vascular cells is a fundamental determinant of vasomotor tone, particularly in resistance arteries where electrical signaling ensures tissue perfusion and pressure regulation. While vascular smooth muscle cell (VSMC) electrophysiology is well studied, the contribution of endothelial cells (ECs)—including Vm dynamics, calcium signaling, and transcriptional adaptability—remains less defined. In pressurized mesenteric arteries, both VSMCs and ECs exhibited heterogeneous Vm (−70 to −40 mV). Endothelial removal collapsed this heterogeneity and shifted smooth muscle cells to depolarized values, establishing the endothelium as a key determinant of resting potential and vasomotor activity. In culture, isolated ECs were depolarized (∼−25 mV), disorganized, and failed to hyperpolarize or elevate calcium with acetylcholine or the KCa activator SKA-31. In contrast, confluent, coupled ECs displayed more negative Vm (−65 to −40 mV) and robust hyperpolarization with synchronized calcium waves and effective responses to KCa activation. This hyperpolarization is a fundamental mechanism of endothelium-dependent hyperpolarization (EDH) underlying vasomotor regulation. These states were reinforced by transcriptional upregulation of FOXO3, MEF2C, KCa2.3, KCa3.1, and Kir2.1, as shown by RNAscope in situ hybridization. Connexin dependence was confirmed by gap junction blockers (carbenoxolone, oleamide), which suppressed hyperpolarized subpopulations and reduced ion channel and transcription factor transcripts. By contrast, Cx43 hemichannel blockade (Gap19) or Cx43MK4 ECs—where hemichannel activity is impaired but gap junctional coupling preserved—did not depolarize Vm, indicating gap junctions, not hemichannels, sustain electrical identity. Importantly, TRPV4-Cx43 clustering, a critical EDH component, was reorganized at borders only under coupled conditions, underscoring that coupling governs both transcriptional programs and spatial organization of signaling complexes. Together, these findings establish coupling as a master regulator of endothelial electrical identity, transcriptional programming, and EDH machinery. R01HL173018, AHA CDA 932684, AHA 23DIVSUP1054931.
Burboa et al. (Sun,) studied this question.