Cha Seung-Kuy, Wu Tao, Huang Chou-Long
Department of Medicine (Division of Nephrology), UT Southwestern Medical Center, Dallas, Texas 75390-8856, USA.
Am J Physiol Renal Physiol. 2008 May;294(5):F1212-21. doi: 10.1152/ajprenal.00007.2008. Epub 2008 Feb 27.
Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial Ca(2+) reabsorption in kidney. Many hormones alter renal Ca(2+) reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal Ca(2+) reabsorption by PTH.
瞬时受体电位香草酸亚型5（TRPV5）构成了肾脏上皮细胞跨上皮钙（Ca2+）重吸收的顶端进入途径。许多激素至少部分通过调节TRPV5来改变肾脏Ca2+重吸收。磷脂酶C偶联激素对TRPV5急性调节的机制很大程度上尚不清楚。在此，我们发现蛋白激酶C（PKC）激活剂1-油酰基-乙酰基-sn-甘油（OAG）增加了培养细胞中TRPV5的电流密度和表面丰度。用特异性PKC抑制剂预孵育可阻止OAG介导的TRPV5增加。与显性负性发动蛋白共表达增加了基础TRPV5电流密度，并阻止了OAG引起的增加。用小干扰RNA（siRNA）敲低小窝蛋白-1可阻止OAG引起的TRPV5增加。相反，敲低网格蛋白重链没有影响。OAG对源自小窝蛋白-1基因敲除小鼠的小窝蛋白-1缺失细胞中表达的TRPV5没有影响。强制表达重组小窝蛋白-1可恢复OAG对小窝蛋白-1基因敲除细胞中TRPV5的调节。TRPV5的丝氨酸-299和/或丝氨酸-654（PKC磷酸化的共有残基）突变消除了OAG的调节作用。甲状旁腺激素（PTH）增加了共表达TRPV5和1型PTH受体的细胞中的TRPV5电流密度。PKC抑制剂、丝氨酸-299/丝氨酸-654突变或敲低小窝蛋白-1可阻止PTH引起的增加。因此，TRPV5经历组成型小窝介导的内吞作用。PKC激活通过抑制内吞作用增加TRPV5的细胞表面丰度。PKC的这种调节机制可能有助于PTH对TRPV5和肾脏Ca2+重吸收的急性刺激。
