Godoy Alejandro, Ormazabal Valeska, Moraga-Cid Gustavo, Zúñiga Felipe A, Sotomayor Paula, Barra Valeria, Vasquez Osmán, Montecinos Viviana, Mardones Lorena, Guzmán Catherine, Villagrán Marcelo, Aguayo Luis G, Oñate Sergio A, Reyes Alejandro M, Cárcamo Juan G, Rivas Coralia I, Vera Juan Carlos
Departamento de Fisiopatología, Universidad de Concepción, Concepción, Casilla 160C, Chile.
J Biol Chem. 2007 Jan 5;282(1):615-24. doi: 10.1074/jbc.M608300200. Epub 2006 Sep 29.
We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter.
我们对人源钠-抗坏血酸转运体SVCT2进行了特性描述，并建立了一个转运循环的基本模型，该模型对其作为钠依赖性转运体的当前观点提出了挑战。SVCT2的特性受到Ca(2+)/Mg(2+)以及钠与抗坏血酸之间相互功能相互作用的调节，这种相互作用定义了底物结合顺序和转运化学计量。钠以协同方式增加抗坏血酸的转运速率，降低转运K(m)而不影响V(max)，从而将转运体的低亲和力形式转变为高亲和力转运体。相反，抗坏血酸以双峰且浓度依赖性的方式影响钠的协同性，在低和高抗坏血酸浓度下均不存在协同性。我们的数据与以2:1的钠:抗坏血酸化学计量和Na(+):抗坏血酸:Na(+)类型的底物结合顺序为特征的转运循环一致。然而，SVCT2不是电生的。SVCT2的功能表现出对Ca(2+)/Mg(2+)的绝对需求，两种阳离子通过增加转运V(max)而不影响转运K(m)或钠协同性，将转运体从无活性构象转变为活性构象。我们的数据表明，SVCT2可能在多种具有特征性质的状态之间切换，包括在没有Ca(2+)/Mg(2+)时的无活性构象。至少可以设想三种活性状态，包括钠浓度低于20 mM时的低亲和力构象以及钠浓度升高时的两种高亲和力构象，其钠协同性受抗坏血酸调节。因此,SVCT2是一种Ca(2+)/Mg(2+)依赖性转运体。
