Cation Interactions and Membrane Potential Induce Conformational Changes in NaPi-IIb
Cation Interactions and Membrane Potential Induce Conformational Changes in NaPi-IIb
复制标题
DOI：
10.1016/j.bpj.2016.07.025
复制
发表时间：
2016-09-06
影响因子：
3.4
通讯作者：
Forster, Ian C.
中科院分区：
文献类型：
--
作者：
Patti, Monica;Fenollar-Ferrer, Cristina;Forster, Ian C.
SLC34 家族的 Na+ 偶联磷酸盐协同转运蛋白的电压依赖性源于蛋白质固有电荷的位移以及响应跨膜电场变化的一个 Na+ 离子的结合/释放。 Na1 位点阳离子的候选配位残基先前通过使用二羧酸转运蛋白 VcINDY 的 X 射线结构作为模板的结构建模进行了预测，并通过功能研究得到了证实。 Na1 的突变导致稳态和前稳态特征的改变，这些特征应该反映在膜电位变化引起的构象变化中。为了通过功能分析检验这一假设，构建了比目鱼 SLC34A2 蛋白的双突变体，其中包含一个 Na1 位点扰动突变以及用于荧光团标记的取代半胱氨酸，如非洲爪蟾卵母细胞中表达的那样。突变的位置被映射到比目鱼蛋白的同源模型上。通过稳态、稳态前和荧光测定来表征诱变的效果。在先前确定的三个位置处解析了响应膜电位阶跃的荧光强度（Delta F）的变化。这些荧光数据证实了 Na1 扰动时前稳态动力学的改变，并且进一步表明了相应荧光团微环境的伴随变化，如电压依赖性和 Delta F 时间过程的变化所证明的那样。此外，碘化物猝灭实验表明荧光团微环境的水性取决于膜电位。这些发现提供了令人信服的证据，证明膜电位和阳离子相互作用会引起蛋白质的大规模结构重排。
Voltage-dependence of Na+-coupled phosphate cotransporters of the SLC34 family arises from displacement of charges intrinsic to the protein and the binding/release of one Na+ ion in response to changes in the transmembrane electric field. Candidate coordination residues for the cation at the Na1 site were previously predicted by structural modeling using the x-ray structure of dicarboxylate transporter VcINDY as template and confirmed by functional studies. Mutations at Na1 resulted in altered steady-state and presteady-state characteristics that should be mirrored in the conformational changes induced by membrane potential changes. To test this hypothesis by functional analysis, double mutants of the flounder SLC34A2 protein were constructed that contain one of the Na1-site perturbing mutations together with a substituted cysteine for fluorophore labeling, as expressed in Xenopus oocytes. The locations of the mutations were mapped onto a homology model of the flounder protein. The effects of the mutagenesis were characterized by steady-state, presteady-state, and fluorometric assays. Changes in fluorescence intensity (Delta F) in response to membrane potential steps were resolved at three previously identified positions. These fluorescence data corroborated the altered presteady-state kinetics upon perturbation of Na1, and furthermore indicated concomitant changes in the microenvironment of the respective fluorophores, as evidenced by changes in the voltage dependence and time course of Delta F. Moreover, iodide quenching experiments indicated that the aqueous nature of the fluorophore microenvironment depended on the membrane potential. These findings provide compelling evidence that membrane potential and cation interactions induce significant large-scale structural rearrangements of the protein.