Renouncing electroneutrality is not free of charge:: Switching on electrogenicity in a Na+-coupled phosphate cotransporter
Renouncing electroneutrality is not free of charge:: Switching on electrogenicity in a Na+-coupled phosphate cotransporter
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DOI：
10.1073/pnas.0505882102
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发表时间：
2005-08-30
影响因子：
11.1
通讯作者：
Forster, IC
中科院分区：
文献类型：
--
作者：
Bacconi, A;Virkki, LV;Forster, IC
肾IIa型Na+偶联无机磷酸盐（Pi）协同转运蛋白（NaPi-IIa）以产电方式介导二价Pi转运，而肾IIc型同种型（NaPi-IIc）是电中性的，但它与NaPi-IIa显示出高度的序列同一性。双摄取（（32）p（i）/Na-22）测定证实，NaPi-IIc显示Na+偶联的Pi共转运，其化学计量比为2：1（Na+：Pi），而NaPi-IIa为3：1。这一发现表明，与NaPi-IIc相比，NaPi-IIa的产电性来自于额外的Na+离子的相互作用。为了确定负责异构体之间的功能差异的分子元件，我们使用嵌合体和氨基酸置换方法。NaPi-IIa和NaPi-IIc构建的嵌合体的转运活性表明，前6个跨膜结构域内的残基是NaPi-IIa的产电性所必需的。产电和电中性异构体之间的序列比较显示，在电荷和极性的残基聚集在三个领域，其中之一，包括预测的第三跨膜结构域的一部分的差异。在此，用NaPi-IIc中的NaPi-Ila等同物（S189 A、S191 A和G195 D）取代三个残基导致显示1：1电荷/P-i偶联、3：1 Na+：P-i化学计量和类似于前稳态弛豫的瞬态电流的转运蛋白。突变体的较弱的电压依赖性和10倍的低表观Pi亲和力相比，NaPi-IIa表明，其他残基的NaPi-IIa动力学指纹的存在。我们的研究结果表明，通过最小数量的侧链取代，我们可以实现从电中性到产电协同转运功能的切换，伴随着一个共底物相互作用位点的出现。
Renal type IIa Na+-coupled inorganic phosphate (Pi) cotransporters (NaPi-IIa) mediate divalent Pi transport in an electrogenic manner, whereas the renal type IIc isoform (NaPi-IIc) is electroneutral, yet it shows high sequence identity with NaPi-IIa. Dual uptake ((32)p(i)/Na-22) assays confirmed that NaPi-IIc displayed Na+-coupled Pi cotransport with a 2:1 (Na+:Pi) stoichiometry compared with 3:1 established for NaPi-IIa. This finding suggested that the electrogenicity of NaPi-IIa arises from the interaction of an additional Na+ ion compared with NaPi-IIc. To identify the molecular elements responsible for the functional difference between isoforms, we used chimera and amino acid replacement approaches. Transport activity of chimeras constructed with NaPi-IIa and NaPi-IIc indicated that residues within the first six transmembrane domains were essential for the electrogenicity of NaPi-IIa. Sequence comparison between electrogenic and electroneutral isoforms revealed differences in the charge and polarity of residues clustered in three areas, one of which included part of the predicted third transmembrane domain. Here, substitution of three residues with their NaPi-Ila equivalents in NaPi-IIc (S189A, S191A, and G195D) resulted in a transporter that displayed a 1:1 charge/P-i coupling, a 3:1 Na+:P-i stoichiometry, and transient currents that resembled pre-steady-state relaxations. The mutant's weaker voltage dependency and 10-fold lower apparent Pi affinity compared with NaPi-Ila indicated that other residues important for the NaPi-Ila kinetic fingerprint exist. Our findings demonstrate that, through a minimal number of side chain substitutions, we can effect a switch from electroneutral to electrogenic cotransporter function, concomitant with the appearance of a cosubstrate interaction site.