Substrate interactions of the electroneutral Na+-coupled inorganic phosphate cotransporter (NaPi-IIc)
Substrate interactions of the electroneutral Na+-coupled inorganic phosphate cotransporter (NaPi-IIc)
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DOI：
10.1113/jphysiol.2009.175596
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发表时间：
2009-09-01
影响因子：
5.5
通讯作者：
Forster, Ian C.
中科院分区：
文献类型：
--
作者：
Ghezzi, Chiara;Murer, Heini;Forster, Ian C.
SLC34 溶质载体家族包括生电 NaPi-IIa/b 和电中性 NaPi-IIc，其 Na+ : P-i 共转运化学计量分别为 3:1 和 2:1。我们之前提出，NaPi-IIc 缺乏为产电异构体假设的三个 Na+ 相互作用位点之一，但与 NaPi-IIa/b 不同，其底物结合顺序尚未确定。通过在爪蟾卵母细胞中表达 NaPi-IIc，同位素流入和流出测定得出的结果与 Na+ 是第一个和最后一个结合的底物一致。为了进一步研究底物相互作用，我们应用了基于荧光测定的技术，该技术使用荧光团的位点特异性标记来表征底物诱导的构象变化。 NaPi-IIc 的第三个细胞外环中引入了一种新的 Cys，可以用报告荧光团 (MTS-TAMRA) 进行标记。虽然标记导致了共转运的抑制，如之前报道的产电亚型，但荧光的变化是由在不存在 P-i 的情况下细胞外 Na+ 浓度的变化以及在存在 Na+ 的情况下细胞外 P-i 浓度的变化引起的。这些数据与 P-32 摄取数据相结合，也支持 Na+ 是第一个相互作用的底物的结合方案。此外，荧光测定法的表观 P-i 亲和力与 P-32 摄取的表观亲和力一致，证实了荧光测定技术对于电中性载体动力学研究的适用性。荧光数据分析表明，与产电 NaPi-IIb 一样，2 个 Na+ 离子在 P-i 结合之前与 NaPi-IIc 协同相互作用，这意味着只有其中一个发生了易位。这一结果提供了令人信服的证据，证明 SLC34 蛋白具有共同的底物相互作用基序，并且共转运和底物结合化学计量不一定相同。
The SLC34 solute carrier family comprises the electrogenic NaPi-IIa/b and the electroneutral NaPi-IIc, which display Na+ : P-i cotransport stoichiometries of 3 : 1 and 2 : 1, respectively. We previously proposed that NaPi-IIc lacks one of the three Na+ interaction sites hypothesised for the electrogenic isoforms, but, unlike NaPi-IIa/b, its substrate binding order is undetermined. By expressing NaPi-IIc in Xenopus oocytes, isotope influx and efflux assays gave results consistent with Na+ being the first and last substrate to bind. To further investigate substrate interactions, we applied a fluorometry-based technique that uses site-specific labelling with a fluorophore to characterize substrate-induced conformational changes. A novel Cys was introduced in the third extracellular loop of NaPi-IIc that could be labelled with a reporter fluorophore (MTS-TAMRA). Although labelling resulted in suppression of cotransport as previously reported for the electrogenic isoforms, changes in fluorescence were induced by changes in extracellular Na+ concentration in the absence of P-i and by changes in extracellular P-i concentration in presence of Na+. These data, combined with P-32 uptake data, also support a binding scheme in which Na+ is the first substrate to interact. Moreover, the apparent P-i affinity from fluorometry agreed with that from P-32 uptake, confirming the applicability of the fluorometric technique for kinetic studies of electroneutral carriers. Analysis of the fluorescence data showed that like the electrogenic NaPi-IIb, 2 Na+ ions interact cooperatively with NaPi-IIc before P-i binding, which implies that only one of these is translocated. This result provides compelling evidence that SLC34 proteins share common motifs for substrate interaction and that cotransport and substrate binding stoichiometries are not necessarily equivalent.