Montgomery Darrice S, Yu Ling, Ghazi Zinah M, Thai Tiffany L, Al-Khalili Otor, Ma He-Ping, Eaton Douglas C, Alli Abdel A
Department of Physiology and Functional Genomics and Department of Medicine Division of Nephrology, Hypertension, and Renal Transplantation, University of Florida College of Medicine, Gainesville, Florida.
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China; and.
Am J Physiol Cell Physiol. 2017 Jul 1;313(1):C42-C53. doi: 10.1152/ajpcell.00244.2016. Epub 2017 May 3.
We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins, we show that MARCKS, but not the ENaC subunits, are a substrate of calpain-2 in the presence of Ca Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light-density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca mobilization and calpain-2 inhibition decrease the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch-clamp recordings and transepithelial current measurements. These results suggest that calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent regulation of ENaC activity in the kidney.
我们之前证明了富含肉豆蔻酰化丙氨酸的蛋白激酶C底物（MARCKS）在阴离子磷脂磷酸依赖性调节上皮钠通道（ENaC）中作为衔接蛋白的作用。MARCKS和ENaC均受蛋白水解作用调节。钙蛋白酶是一族广泛表达的细胞内钙依赖性半胱氨酸蛋白酶，参与信号转导。在此，我们研究钙蛋白酶-2在调节培养的肾上皮细胞和小鼠肾脏中的MARCKS和ENaC的作用。使用重组融合蛋白，我们发现在有钙的情况下，MARCKS是钙蛋白酶-2的底物，而ENaC亚基不是。对钙蛋白酶-2的药理学抑制改变了来自小鼠皮质集合管细胞裂解物的轻密度蔗糖梯度级分中MARCKS蛋白的表达。在培养的肾细胞中可检测到MARCKS的钙蛋白酶依赖性裂解产物。钙动员和钙蛋白酶-2抑制降低了ENaC与MARCKS之间的结合。如单通道膜片钳记录和跨上皮电流测量所示，钙蛋白酶-2的抑制降低了ENaC活性。这些结果表明，MARCKS的钙蛋白酶-2蛋白水解作用促进其在质膜上与脂质和ENaC的相互作用，从而实现肾脏中磷脂酰肌醇4,5-二磷酸（PIP2）依赖性的ENaC活性调节。
