Wang Lingyun, Holmes Ross P, Peng Ji-Bin
Division of Nephrology, Department of Medicine, Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Department of Urology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Biochem Biophys Res Commun. 2017 Oct 21;492(3):362-367. doi: 10.1016/j.bbrc.2017.08.102. Epub 2017 Aug 25.
TRPV5 is a Ca-selective channel that plays a key role in the reabsorption of Ca ions in the kidney. Recently, a rare L530R variation (rs757494578) of TRPV5 was found to be associated with recurrent kidney stones in a founder population. However, it was unclear to what extent this variation alters the structure and function of TRPV5. To evaluate the function and expression of the TRPV5 variant, Ca uptake in Xenopus oocytes and western blot analysis were performed. The L530R variation abolished the Ca uptake activity of TRPV5 in Xenopus oocytes. The variant protein was expressed with drastic reduction in complex glycosylation. To assess the structural effects of this L530R variation, TRPV5 was modeled based on the crystal structure of TRPV6 and molecular dynamics simulations were carried out. Simulation results showed that the L530R variation disrupts the hydrophobic interaction between L530 and L502, damaging the secondary structure of transmembrane domain 5. The variation also alters its interaction with membrane lipid molecules. Compared to the electroneutral L530, the positively charged R530 residue shifts the surface electrostatic potential towards positive. R530 is attracted to the negatively charged phosphate group rather than the hydrophobic carbon atoms of membrane lipids. This shifts the pore helix where R530 is located and the D542 residue in the Ca-selective filter towards the surface of the membrane. These alterations may lead to misfolding of TRPV5, reduction in translocation of the channel to the plasma membrane and/or impaired Ca transport function of the channel, and ultimately disrupt TRPV5-mediated Ca reabsorption.
瞬时受体电位香草酸亚型5（TRPV5）是一种对钙离子具有选择性的通道，在肾脏中钙离子的重吸收过程中发挥关键作用。最近，在一个奠基者群体中发现，TRPV5的一种罕见的L530R变异（rs757494578）与复发性肾结石有关。然而，尚不清楚这种变异在多大程度上改变了TRPV5的结构和功能。为了评估TRPV5变异体的功能和表达情况，进行了非洲爪蟾卵母细胞的钙摄取实验以及蛋白质免疫印迹分析。L530R变异消除了非洲爪蟾卵母细胞中TRPV5的钙摄取活性。该变异蛋白表达时复合糖基化显著减少。为了评估L530R变异的结构效应，基于TRPV6的晶体结构对TRPV5进行建模，并开展了分子动力学模拟。模拟结果表明，L530R变异破坏了L530与L502之间的疏水相互作用，损害了跨膜结构域5的二级结构。该变异还改变了其与膜脂分子的相互作用。与电中性的L530相比，带正电荷的R530残基使表面静电势向正方向偏移。R530被带负电荷的磷酸基团吸引，而非膜脂的疏水碳原子。这使得R530所在的孔螺旋以及钙选择性过滤器中的D542残基向膜表面移动。这些改变可能导致TRPV5错误折叠，通道向质膜转运减少和/或通道的钙转运功能受损，最终破坏TRPV5介导的钙重吸收。
