Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel, hHV1.
Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel, hHV1.
复制标题
DOI：
10.1085/jgp.201511456
复制
发表时间：
2015-11
期刊：
影响因子：
--
通讯作者：
DeCoursey TE
中科院分区：
文献类型：
--
作者：
Cherny VV;Morgan D;Musset B;Chaves G;Smith SM;DeCoursey TE
电压门控质子通道的电压传感域中独特存在的保守色氨酸对于四个通道定义特性至关重要。定义电压门控质子通道 (HV1) 的“特征序列”的一部分是与 S4 跨膜螺旋中第二个 Arg 相邻的色氨酸残基：RxWRxxR，它在所有高可信度 HV1 基因中都完全保守。用 Ala、Ser 或 Phe 替换人 HV1 (hHV1) 中的 Trp207 可促进门控，使通道打开速度加快 100 倍，关闭速度加快 30 倍。突变体通道在比野生型 (WT) 通道更负的电压下打开，表明在 WT 通道中，Trp 倾向于关闭状态。 WT 通道打开的阿伦尼乌斯活化能 Ea 从 30-38 kcal/mol 降至 22 kcal/mol，证实 Trp207 在关闭和开放 hHV1 之间建立了主要能量屏障。 Trp207 和 Arg211 之间的阳离子-π 相互作用明显锁定了通道关闭。 Trp207 突变体在 pHo >8.0 时失去质子选择性。最后，依赖于跨膜 pH 梯度的门控（ΔpH 依赖性门控）是 HV1 的一个普遍特征，对其生物学功能至关重要，但这一特征受到了损害。在 WT hHV1 中，ΔpH 依赖性门控在 pHi 或 pHo 8 以上饱和，与交替访问内部和外部溶液的单个 pH 传感器一致。然而，饱和度的发生与 ΔpH 无关，表明存在不同的内部和外部 pH 传感器。在 Trp207 突变体中，ΔpH 依赖性门控在较低 pHo 下饱和，但在较低 pHi 下不饱和。 Trp207 突变选择性地改变 pHo 传感，进一步支持了不同的内部和外部 pH 传感器的存在。来自单细胞物种 Karlodinium veneficum 和 Emiliania huxleyi 的 HV1 的类似突变产生了大致相似的结果。在所有三个物种的 HV1 中，ΔpH 依赖性门控的饱和发生在相同的 pHo 和 pHi 下，表明相同或相似的基团参与 pH 传感。因此，Trp 具有四种特性：缓慢的通道打开、高度依赖于温度的门控动力学、质子选择性和依赖于 ΔpH 的门控。
A conserved tryptophan uniquely present in the voltage-sensing domain of voltage-gated proton channels is surprisingly crucial to four channel-defining properties. Part of the “signature sequence” that defines the voltage-gated proton channel (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence HV1 genes. Replacing Trp207 in human HV1 (hHV1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hHV1. Cation–π interaction between Trp207 and Arg211 evidently latches the channel closed. Trp207 mutants lost proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of HV1 that is essential to its biological functions, was compromised. In the WT hHV1, ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in HV1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in HV1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.