Wang Xiaoyu, Rusinova Radda, Gregorio G Glenn, Boudker Olga
Department of Physiology & Biophysics, Weill Cornell Medicine, New York, New York, USA.
Department of Physiology & Biophysics, Weill Cornell Medicine, New York, New York, USA.
J Biol Chem. 2024 Dec;300(12):107955. doi: 10.1016/j.jbc.2024.107955. Epub 2024 Nov 2.
Glutamate is the main excitatory transmitter in the mammalian central nervous system; glutamate transporters keep the synaptic glutamate concentrations at bay for normal brain function. Arachidonic acid (AA), docosahexaenoic acid, and other unsaturated fatty acids modulate glutamate transporters in cell- and tissue slices-based studies. Here, we investigated their effect and mechanism using a purified archaeal glutamate transporter homolog reconstituted into the lipid membranes. AA, docosahexaenoic acid, and related fatty acids irreversibly inhibited the sodium-dependent concentrative substrate uptake into lipid vesicles within the physiologically relevant concentration range. In contrast, AA did not inhibit amino acid exchange across the membrane. The length and unsaturation of the aliphatic tail affect inhibition, and the free carboxylic headgroup is necessary. The inhibition potency did not correlate with the fatty acid effects on the bilayer deformation energies. AA does not affect the conformational dynamics of the protein, suggesting it does not inhibit structural transitions necessary for transport. Single-transporter and membrane voltage assays showed that AA and related fatty acids mediate cation leak, dissipating the driving sodium gradient. Thus, such fatty acids can act as cation ionophores, suggesting a general modulatory mechanism of membrane channels and ion-coupled transporters.
谷氨酸是哺乳动物中枢神经系统中的主要兴奋性神经递质；谷氨酸转运体可控制突触谷氨酸浓度，以维持大脑正常功能。在基于细胞和组织切片的研究中，花生四烯酸（AA）、二十二碳六烯酸及其他不饱和脂肪酸可调节谷氨酸转运体。在此，我们利用一种纯化的古细菌谷氨酸转运体同源物重构到脂质膜中，研究了它们的作用及机制。在生理相关浓度范围内，AA、二十二碳六烯酸及相关脂肪酸不可逆地抑制了脂质囊泡中依赖钠的浓缩性底物摄取。相比之下，AA并不抑制跨膜氨基酸交换。脂肪链尾部的长度和不饱和度会影响抑制作用，游离羧基头部基团是必需的。抑制效力与脂肪酸对双层膜变形能的影响无关。AA不影响蛋白质的构象动力学，这表明它不会抑制转运所需的结构转变。单转运体和膜电压测定表明，AA及相关脂肪酸介导阳离子泄漏，消耗驱动钠梯度。因此，这类脂肪酸可作为阳离子离子载体，提示了一种膜通道和离子偶联转运体的普遍调节机制。
