Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigatedin vitrousing endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.

Significance: Plasma membrane ion channels composed of pore-forming and auxiliary subunits regulate physiological functions in virtually all cell types. A conventional view is that ion channels assemble with their auxiliary subunits prior to surface trafficking of the multiprotein complex. Arterial myocytes express large-conductance Ca ²⁺ -activated potassium (BK) channel α and auxiliary β1 subunits that modulate contractility and blood pressure and flow. The data here show that although most BKα subunits are plasma membrane-resident, β1 subunits are primarily intracellular in arterial myocytes. Nitric oxide, an important vasodilator, stimulates rapid surface trafficking of β1 subunits, which associate with, and activate, BK channels, leading to vasodilation. Thus, we show that rapid auxiliary subunit trafficking is a unique mechanism to control functional surface ion channel activity.

Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α₁-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na⁺ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl ⁻ ) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca ²⁺ )–activated Cl ⁻ currents in endothelial cells from control ( TMEM16A ^fl/fl ) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell–specific knockout of TMEM16A ( TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca ²⁺ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca ²⁺ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca ²⁺ -dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (β1) subunits which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR-enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 μM, we detected BK activation in response to in vivo and in vitro CLR-enrichment of myocytes. While a significant increase in myocyte CLR was achieved within five minutes of CLR in vitro loading, this brief CLR-enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process(es). Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR-enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR-enrichment. Moreover, CLR-enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR-enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.