TRPC6 N338S is a gain-of-function mutant identified in patient with doxorubicin-induced cardiotoxicity.
TRPC6 N338S is a gain-of-function mutant identified in patient with doxorubicin-induced cardiotoxicity.
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DOI：
10.1016/j.bbadis.2022.166505
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发表时间：
2022-07
期刊：
影响因子：
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通讯作者：
T. Lu;Xiaojing Sun;B. Necela;Hon-Chi Lee;N. Norton
中科院分区：
文献类型：
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作者：
T. Lu;Xiaojing Sun;B. Necela;Hon-Chi Lee;N. Norton
典型的瞬时受体电位基因TRPC6被认为是化疗诱导的充血性心力衰竭的风险基因，但缺乏对特定风险变异的了解。在我们的全基因组关联研究和随后的精细定位之后，在一位接受含蒽环类药物化疗方案并发展为充血性心力衰竭的乳腺癌患者中发现了罕见的TRPC6 N338S错sense突变体。然而，N338S突变体的功能尚未得到检验。利用细胞内Ca2+成像、膜片钳记录和分子对接技术，我们评估了N338S突变体在HEK293细胞和HL-1心肌细胞中异源表达的功能。我们发现，与TRPC6 WT相比，TRPC6 N338S在50 μM 1-油基2-乙酰基-森-甘油（OAG） （TRPC6通道激活剂）下的表达显著增加了细胞内Ca2+水平（[Ca2+]i）和电流密度。0.5 μM阿霉素（DOX）预处理24小时进一步增强了OAG对TRPC6 N338S电流密度和[Ca2+]i的影响，而这些影响被1 μM BI-749327（一种高选择性TRPC6抑制剂）消除。此外，与TRPC6 WT相比，DOX处理显著上调了TRPC6 N338S的mRNA和蛋白表达。分子对接和动力学模拟表明，OAG与TRPC6的孔螺旋结构域、S5和S6结构域构成的口袋结合。然而，N338S突变加强了与OAG的相互作用，从而稳定了OAG- trpc6 N338S复合物，增强了OAG的结合亲和力。我们的研究结果表明，TRPC6 N338S是一种功能获得突变体，可能通过增加心肌细胞中的Ca2+内流和[Ca2+] ix来促进dox诱导的心脏毒性。
The canonical transient receptor potential 6 gene,TRPC6, has been implicated as a putative risk gene for chemotherapy-induced congestive heart failure, but knowledge of specific risk variants is lacking. Following our genome-wide association study and subsequent fine-mapping, a rare missense mutant of TRPC6 N338S, was identified in a breast cancer patient who received anthracycline-containing chemotherapy regiments and developed congestive heart failure. However, the function of N338S mutant has not been examined. Using intracellular Ca2+imaging, patch clamp recording and molecular docking techniques, we assessed the function of N338S mutant heterologously expressed in HEK293 cells and HL-1 cardiac cells. We found that expression of TRPC6 N338S significantly increased intracellular Ca2+levels ([Ca2+]i) and current densities in response to 50 μM 1-oleoyl 2-acetyl-sn-glycerol (OAG), an activator of TRPC6 channels, compared to those of TRPC6 WT. A 24-h pretreatment with 0.5 μM doxorubicin (DOX) further potentiated the OAG effects on TRPC6 N338S current densities and [Ca2+]i, and these effects were abolished by 1 μM BI-749327, a highly selective TRPC6 inhibitor. Moreover, DOX treatment significantly upregulated the mRNA and protein expressions of TRPC6 N338S, compared to those of TRPC6 WT. Molecular docking and dynamics simulation showed that OAG binds to the pocket constituted by the pore-helix, S5 and S6 domains of TRPC6. However, the N338S mutation strengthened the interaction with OAG, therefore stabilizing the OAG-TRPC6 N338S complex and enhancing OAG binding affinity. Our results indicate that TRPC6 N338S is a gain-of-function mutant that may contribute to DOX-induced cardiotoxicity by increasing Ca2+influx and [Ca2+]iin cardiomyocytes.