Data_Sheet_1_TRPV4 Complexes With the Na+/Ca2+ Exchanger and IP3 Receptor 1 to Regulate Local Intracellular Calcium and Tracheal Tension in Mice.PDF

Intracellular Ca2+ is critical for regulating airway smooth muscle (ASM) tension. A rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) of ASM cells is crucial for modulating the intensity and length of the ASM contraction. Because this rapid increase in [Ca2+]i largely depends on the balance between Ca2+ released from intracellular Ca2+ stores and extracellular Ca2+ entry, exploring the mechanisms mediating Ca2+ transport is critical for understanding ASM contractility and the pathogenesis of bronchial contraction disorders. Transient receptor potential vanilloid 4 (TRPV4) is a highly Ca2+-permeable non-selective cation channel that mediates Ca2+ influx to increase [Ca2+]i, which then directly or indirectly regulates the contraction and relaxation of ASM. The [Ca2+]i returns to basal levels through several uptake and extrusion pumps, such as the sarco(endo)plasmic reticulum Ca2+ ATPase and inositol 1,4,5-trisphosphate receptors (IP3Rs), the plasmalemmal Ca2+ ATPase, and the plasma membrane Na+/Ca2+ exchanger (NCX). Thus, to further understand ASM tension regulation in normal and diseased tissue, the present study examined whether an interaction exists among TRPV4, IP3Rs, and NCX. The TRPV4-specific and potent agonist GSK1016790A increased [Ca2+]i in mouse ASM cells, an effect that was completely blocked by the TRPV4-specific antagonist HC067047. However, GSK1016790A induced relaxation in mouse tracheal rings precontracted with carbachol in vitro. To determine the mechanism underlying this TRPV4-induced relaxation of ASM, we blocked specific downstream molecules. We found that the GSK1016790A-induced relaxation was abolished by the NCX inhibitors KB-R7943 and LiCl but not by specific inhibitors of the Ca2+-activated large-, intermediate-, or small-conductance K+ channels (BKCa, IK, and SK3, respectively). The results of co-immunoprecipitation (co-IP) assays showed an interaction of TRPV4 and IP3R1 with NCXs. Taken together, these findings support a physical and functional interaction of TRPV4 and IP3R1 with NCXs as a novel TRPV4-mediated Ca2+ signaling mechanism and suggest a potential target for regulation of ASM tension and treatment of respiratory diseases, especially tracheal spasm.

细胞内钙离子（Intracellular Ca²⁺）在调控气道平滑肌（airway smooth muscle, ASM）张力中发挥关键作用。气道平滑肌细胞内钙离子浓度（[Ca²⁺]i）的快速升高，对于调节气道平滑肌收缩的强度与时长至关重要。由于[Ca²⁺]i的快速升高主要依赖于细胞内钙库释放的钙离子与细胞外钙离子内流之间的平衡，因此探究介导钙离子转运的机制，对于理解气道平滑肌收缩功能以及支气管收缩紊乱的发病机制具有重要意义。瞬时受体电位香草酸亚型4（Transient receptor potential vanilloid 4, TRPV4）是一种高钙离子通透性的非选择性阳离子通道，可介导钙离子内流以升高[Ca²⁺]i，进而直接或间接调控气道平滑肌的收缩与舒张。细胞内[Ca²⁺]i可通过多种摄取与排出泵回到基础水平，例如肌浆（内质）网钙离子ATP酶（sarco(endo)plasmic reticulum Ca²⁺ ATPase）、1,4,5-三磷酸肌醇受体（inositol 1,4,5-trisphosphate receptors, IP3Rs）、质膜钙离子ATP酶以及质膜Na⁺/Ca²⁺交换体（plasma membrane Na⁺/Ca²⁺ exchanger, NCX）。因此，为进一步阐明正常与病变组织中气道平滑肌张力的调控机制，本研究探究了TRPV4、IP3Rs与NCX之间是否存在相互作用。TRPV4特异性强效激动剂GSK1016790A可升高小鼠气道平滑肌细胞内的[Ca²⁺]i，该效应可被TRPV4特异性拮抗剂HC067047完全阻断。然而，GSK1016790A可使预先经卡巴胆碱预收缩的小鼠气管环在体外发生舒张。为明确该TRPV4介导的气道平滑肌舒张的潜在机制，我们对特定下游分子进行了阻断实验。结果发现，GSK1016790A诱导的舒张效应可被NCX抑制剂KB-R7943和氯化锂（LiCl）消除，但不受钙激活大电导、中电导以及小电导钾通道（分别为BKCa、IK和SK3）的特异性抑制剂的影响。免疫共沉淀（co-immunoprecipitation, co-IP）实验结果显示，TRPV4与IP3R1均可与NCX发生相互作用。综上，本研究结果表明，TRPV4与IP3R1和NCX之间存在物理与功能上的相互作用，这是一种新型的TRPV4介导的钙离子信号转导机制，同时提示该通路可作为调控气道平滑肌张力以及治疗呼吸系统疾病（尤其是气管痉挛）的潜在靶点。