Shafqat S, Velaz-Faircloth M, Henzi V A, Whitney K D, Yang-Feng T L, Seldin M F, Fremeau R T
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Mol Pharmacol. 1995 Aug;48(2):219-29.
L-Proline fulfills several of the classic criteria used to identify amino acid neurotransmitters, including the presence of a high affinity, Na(+)- (and Cl-)-dependent synaptosomal transport process and the Ca(2+)-dependent release of exogenously loaded radiolabeled L-proline from brain slices and synaptosomes after K(+)-induced depolarization. However, studies to define the role of L-proline in discrete pathways in the mammalian brain have been precluded by the inability to block its biosynthesis or high affinity transport in nervous tissue. We report the molecular cloning, functional expression, and chromosomal localization of a human brain-specific high affinity L-proline transporter (hPROT). The pharmacological specificity, kinetic properties, and ionic requirements of hPROT clearly distinguish this carrier from the other Na(+)-dependent plasma membrane carriers that transport L-proline. Multiple tissue Northern blot analysis revealed a prominent approximately 4-kb mRNA transcript in human brain tissue, whereas no specific hybridizing species were detected in peripheral tissue. An antipeptide antiserum directed against the carboxy-terminus of the predicted hPROT protein identified a single, broad immunoreactive protein of 68 kDa on immunoblots of synaptosomal membranes from various human brain regions. In contrast, no specific labeling was detected on immunoblots of membranes from human liver, kidney, or heart. A differential distribution of hPROT mRNA and protein was observed in the human corpus striatum, consistent with the hypothesis that the hPROT protein is synthesized in neuronal cell bodies in an extrastriatal location and axonally transported to the corpus striatum. These findings warrant the consideration of a synaptic regulatory role for this transporter and its presumed natural substrate, L-proline, in the mammalian central nervous system.
L-脯氨酸符合用于鉴定氨基酸神经递质的多项经典标准，包括存在高亲和力、依赖Na⁺（和Cl⁻）的突触体转运过程，以及在K⁺诱导的去极化后，从脑片和突触体中外源加载的放射性标记L-脯氨酸的Ca²⁺依赖性释放。然而，由于无法阻断其在神经组织中的生物合成或高亲和力转运，关于确定L-脯氨酸在哺乳动物脑离散通路中作用的研究受到了阻碍。我们报告了一种人脑特异性高亲和力L-脯氨酸转运体（hPROT）的分子克隆、功能表达及染色体定位。hPROT的药理学特异性、动力学特性和离子需求明显将该载体与其他转运L-脯氨酸的依赖Na⁺的质膜载体区分开来。多组织Northern印迹分析显示，在人脑组织中有一个约4kb的显著mRNA转录本，而在外周组织中未检测到特异性杂交条带。针对预测的hPROT蛋白羧基末端的抗肽抗血清，在来自不同人脑区域的突触体膜免疫印迹上鉴定出一个单一的、68kDa的宽免疫反应性蛋白。相比之下，在人肝、肾或心脏的膜免疫印迹上未检测到特异性标记。在人类纹状体中观察到hPROT mRNA和蛋白的差异分布，这与hPROT蛋白在纹状体以外位置的神经元细胞体中合成并通过轴突运输到纹状体的假说一致。这些发现值得考虑该转运体及其假定的天然底物L-脯氨酸在哺乳动物中枢神经系统中的突触调节作用。
