Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Am J Physiol Cell Physiol. 2012 Oct 15;303(8):C834-42. doi: 10.1152/ajpcell.00171.2012. Epub 2012 Jul 25.
The proton-coupled folate transporter (PCFT-SLC46A1) mediates intestinal folate absorption and folate transport across the choroid plexus, processes defective in hereditary folate malabsorption (HFM). This paper characterizes the functional defect, and the roles of two mutated PCFT residues, associated with HFM (G338R and A335D). The A335D-PCFT and other mutations at this residue result in an unstable protein; when expression of a mutant protein was preserved, function was always retained. The G338R and other charged mutants resulted in an unstable protein; substitutions with small neutral and polar amino acids preserved protein but with impaired function. Pemetrexed and methotrexate (MTX) influx kinetics mediated by the G338C mutant PCFT revealed marked (15- to 20-fold) decreases in K(t) and V(max) compared with wild-type PCFT. In contrast, there was only a small (∼2-fold) decrease in the MTX influx K(i) and an increase (∼3-fold) in the pemetrexed influx K(i) for the G338C-PCFT mutant. Neither a decrease in pH to 4.5, nor an increase to 7.4, restored function of the G338C mutant relative to wild-type PCFT excluding a role for this residue in proton binding or proton coupling. Homology modeling localized the A335 and G338 residues embedded in the 9th transmembrane, consistent with the inaccessibility of the A335C and G338C proteins to MTS reagents. Hence, the loss of intrinsic G338C-PCFT function was due solely to impaired oscillation of the carrier between its conformational states. The data illustrate how alterations in carrier cycling can impact influx K(t) without comparable alterations in substrate binding to the carrier.
质子偶联叶酸转运体（PCFT-SLC46A1）介导肠道叶酸吸收和脉络丛中的叶酸转运，其功能缺陷与遗传性叶酸吸收不良（HFM）有关。本文描述了与 HFM 相关的两个突变 PCFT 残基（G338R 和 A335D）的功能缺陷及其作用。A335D-PCFT 和该残基的其他突变导致蛋白不稳定；当突变蛋白的表达得到保留时，功能总是保留的。G338R 和其他带电荷的突变导致蛋白不稳定；用小的中性和极性氨基酸取代可以保留蛋白，但功能受损。与野生型 PCFT 相比，G338C 突变 PCFT 介导的培美曲塞和氨甲蝶呤（MTX）内流动力学显示 K(t)和 V(max)显著降低（15-20 倍）。相比之下，G338C-PCFT 突变体的 MTX 内流 K(i)仅略有降低（约 2 倍），而培美曲塞内流 K(i)增加（约 3 倍）。G338C 突变体的功能既不能通过降低 pH 值至 4.5 恢复，也不能通过升高至 7.4 恢复，这排除了该残基在质子结合或质子偶联中的作用。同源建模将 A335 和 G338 残基定位于第 9 个跨膜区，与 A335C 和 G338C 蛋白对 MTS 试剂不可接近的情况一致。因此，G338C-PCFT 内在功能的丧失完全是由于载体在构象状态之间的摆动受损所致。数据说明了载体循环的改变如何在不改变载体对底物结合的情况下影响内流 K(t)。
