TMEM16A Ca²⁺-Activated Cl^- Channel Regulates the Proliferation and Migration of Brain Capillary Endothelial Cells.

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca²⁺-activated Cl^- (Cl_Ca) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl^- currents were sensitive to the Cl_Ca channel blockers, 100 μM niflumic acid and 10 μM T16A_inh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of Cl_Ca currents with Cl_Ca channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt). T16A_inh-A01 reduced cell viability in a concentration-dependent manner. Either Cl_Ca channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, Cl_Ca channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to Cl_Ca channel conductance and can regulate both the resting membrane potential and [Ca²⁺]_cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca²⁺-activated Cl^- channels and can regulate both the resting membrane potential and cytosolic Ca²⁺ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.

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