Teng Jinfeng, Loukin Stephen H, Anishkin Andriy, Kung Ching
Laboratory of Molecular and Cell Biology, University of Wisconsin, Madison, WI 53706; Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Laboratory of Molecular and Cell Biology, University of Wisconsin, Madison, WI 53706.
Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):11847-11852. doi: 10.1073/pnas.1613523113. Epub 2016 Oct 3.
We have some generalized physical understanding of how ion channels interact with surrounding lipids but few detailed descriptions on how interactions of particular amino acids with contacting lipids may regulate gating. Here we discovered a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (transient receptor potential vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between the indole of W733 of the TRP helix and the backbone oxygen of L596 secures the helix/linker contact, which acts as a latch maintaining channel closure. The modeled side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model-an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Making this residue less hydrophobic (L596A/G/W/Q/K) reduces open probability [Po; loss-of-function (LOF)], likely due to altered interactions at the polar-nonpolar interface. L596I raises Po [gain-of-function (GOF)], apparently by placing its methyl group further inward and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the levels of α-carbons of H-bonded residues L596 and W733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP helix latch, respectively. These results highlight that L596 lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4.
我们对离子通道如何与周围脂质相互作用有一些一般性的物理理解，但对于特定氨基酸与接触脂质的相互作用如何调节门控，却鲜有详细描述。在此，我们发现了一种氨基酸与内膜脂质之间的结构特异性相互作用，它控制着瞬时受体电位香草酸亚型4（TRPV4）的门控转换。许多阳离子通道利用S4 - S5连接子将刺激传递至门控部位。在TRPV4连接子螺旋的起始处是亮氨酸596。TRP螺旋的W733的吲哚环与L596的主链氧之间形成的氢键确保了螺旋/连接子的接触，其作用如同一个锁扣维持通道关闭。在我们的模型中，L596的模拟侧链与极性 - 非极性界面附近的内膜脂质小叶相互作用，我们通过诱变对这种相互作用进行了探究。我们检测了表达TRPV4的非洲爪蟾卵母细胞在去极化时的外向电流以及表达该通道的酵母细胞的表型。使这个残基疏水性降低（L596A/G/W/Q/K）会降低开放概率[Po；功能丧失（LOF）]，这可能是由于极性 - 非极性界面处的相互作用改变所致。L596I增加了Po[功能获得（GOF）]，显然是通过将其甲基基团进一步向内放置并受到更强的水排斥作用。分子动力学模拟表明，在功能丧失突变体中，形成氢键的残基L596和W733的α - 碳原子水平之间的距离缩短，而在功能获得突变体中则延长，这分别增强或减弱了连接子/TRP螺旋锁扣。这些结果表明，L596与脂质的吸引力在一场拔河比赛中抵消了锁扣键，从而调节TRPV4的Po。
