Notch enhances Ca²⁺ entry by activating calcium-sensing receptors and inhibiting voltage-gated K⁺ channels

Song S, Babicheva A, Zhao T, Ayon RJ, Rodriguez M, Rahimi S, Balistrieri F, Harrington A, Shyy JY, Thistlethwaite PA, Makino A, Yuan JX. Notch enhances Ca²⁺ entry by activating calcium-sensing receptors and inhibiting voltage-gated K⁺ channels. Am J Physiol Cell Physiol 318: C954–C968, 2020. First published March 18, 2020; doi:10.1152/ajpcell.00487.2019.—The increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K⁺ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca²⁺ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca²⁺ influx through receptor-operated and store-operated Ca²⁺ channels (SOC). Upon Ca²⁺ depletion from the SR, STIM forms clusters to mediate store-operated Ca²⁺ entry. Activity of K_V channels, like KCNA5/K_V1.5 and KCNA2/K_V1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca²⁺]cyt by opening voltage-dependent Ca²⁺ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca²⁺ influx through SOC channels. Extracellular Ca²⁺-mediated activation of CaSR increases [Ca²⁺]_cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca²⁺]_cyt. Moreover, CaSR-mediated increase in [Ca²⁺]_cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K⁺ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca²⁺]_cyt by enhancing store-operated Ca²⁺ entry and inhibits KCNA5/K_V1.5 and KCNA2/K_V1.2, ultimately leading to voltage-activated Ca²+ entry.