Modulation of TRPM2 by acidic pH and the underlying mechanisms for pH sensitivity.
Modulation of TRPM2 by acidic pH and the underlying mechanisms for pH sensitivity.
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DOI：
10.1085/jgp.200910254
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发表时间：
2009-12
期刊：
影响因子：
--
通讯作者：
Yue L
中科院分区：
文献类型：
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作者：
Du J;Xie J;Yue L
TRPM2 是一种 Ca2+ 渗透性非选择性阳离子通道，在氧化应激介导的细胞死亡和炎症过程中发挥重要作用。然而，TRPM2在生理和病理条件下如何受到调控尚不完全清楚。在这里，我们报告细胞内和细胞外质子都通过抑制通道门控来阻断 TRPM2。我们证明外部质子阻断 TRPM2，IC50 为 pHo = 5.3，而内部质子抑制 TRPM2，IC50 为 pHi = 6.7。细胞外质子通过降低单通道电导来抑制 TRPM2。我们在通道孔的外前庭确定了三个可滴定残基 H958、D964 和 E994，它们负责 pHo 敏感性。这些残基的突变会降低单通道电导，降低外部 Ca2+ ([Ca2+]o) 亲和力，并抑制 [Ca2+]o 介导的 TRPM2 门控。这些结果支持以下模型：外部质子滴定 H958、D964 和 E994 通过引起通道构象变化和/或通过降低外前庭的局部 Ca2+ 浓度来抑制 TRPM2 门控，从而减少 [Ca2+]o 渗透并抑制 [Ca2+]o 介导的 TRPM2 门控。我们发现细胞内质子通过诱导通道关闭而不改变通道电导来抑制 TRPM2。我们确定位于 S4-S5 连接子 C 末端的 D933 负责细胞内 pH 敏感性。用 Asn933 替换 Asp933 将 IC50 从 pHi = 6.7 更改为 pHi = 5.5。此外，用各种残基取代 Asp933 会导致质子敏感性、细胞内 ADP 核糖/Ca2+ 敏感性和 TRPM2 的门控谱发生显着变化。这些结果表明 D933 不仅对于细胞内 pH 敏感性至关重要，而且对于 TRPM2 通道门控也至关重要。总的来说，我们的研究结果提供了 TRPM2 调节的新机制以及 TRPM2 pH 调节的分子决定因素。酸性 pH 值对 TRPM2 的抑制可能代表了控制 TRPM2 门控及其生理/病理功能的内源机制。
TRPM2 is a Ca2+-permeable nonselective cation channel that plays important roles in oxidative stress–mediated cell death and inflammation processes. However, how TRPM2 is regulated under physiological and pathological conditions is not fully understood. Here, we report that both intracellular and extracellular protons block TRPM2 by inhibiting channel gating. We demonstrate that external protons block TRPM2 with an IC50 of pHo = 5.3, whereas internal protons inhibit TRPM2 with an IC50 of pHi = 6.7. Extracellular protons inhibit TRPM2 by decreasing single-channel conductance. We identify three titratable residues, H958, D964, and E994, at the outer vestibule of the channel pore that are responsible for pHo sensitivity. Mutations of these residues reduce single-channel conductance, decrease external Ca2+ ([Ca2+]o) affinity, and inhibit [Ca2+]o-mediated TRPM2 gating. These results support the following model: titration of H958, D964, and E994 by external protons inhibits TRPM2 gating by causing conformation change of the channel, and/or by decreasing local Ca2+ concentration at the outer vestibule, therefore reducing [Ca2+]o permeation and inhibiting [Ca2+]o-mediated TRPM2 gating. We find that intracellular protons inhibit TRPM2 by inducing channel closure without changing channel conductance. We identify that D933 located at the C terminus of the S4-S5 linker is responsible for intracellular pH sensitivity. Replacement of Asp933 by Asn933 changes the IC50 from pHi = 6.7 to pHi = 5.5. Moreover, substitution of Asp933 with various residues produces marked changes in proton sensitivity, intracellular ADP ribose/Ca2+ sensitivity, and gating profiles of TRPM2. These results indicate that D933 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM2 channel gating. Collectively, our findings provide a novel mechanism for TRPM2 modulation as well as molecular determinants for pH regulation of TRPM2. Inhibition of TRPM2 by acidic pH may represent an endogenous mechanism governing TRPM2 gating and its physiological/pathological functions.