GLT-1 is the main glutamate transporter in the brain and its trafficking controls its availability at the cell surface, thereby shaping glutamatergic neurotransmission under physiological and pathological conditions. Extracellular glutamate is known to trigger ubiquitin-dependent GLT-1 internalization from the surface of the cell to the intracellular compartment, yet here we show that internalization also requires the participation of calcium ions. Consistent with previous studies, the addition of glutamate (1 mM) to mixed primary cultures (containing neurons and astrocytes) promotes GLT-1 internalization, an effect that was suppressed in the absence of extracellular Ca²⁺. The pathways of Ca²⁺ mobilization by astrocytes were analyzed in these mixed cultures using the genetically encoded calcium sensor GCaMP6f. A complex pattern of calcium entry was activated by glutamate, with a dramatic and rapid rise in the intracellular Ca²⁺ concentration partially driven by glutamate transporters, especially in the initial stages after exposure to glutamate. The Na⁺/Ca²⁺ exchanger (NCX) plays a dominant role in this Ca²⁺ mobilization and its blockade suppresses the glutamate induced internalization of GLT-1, both in astrocytes and in a more straightforward experimental system like HEK293 cells transiently transfected with GLT-1. This regulatory mechanism might be relevant to control the amount of GLT-1 transporter at the cell surface in conditions like ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated and they promote rapid Ca²⁺ mobilization.

GLT-1是大脑中主要的谷氨酸转运蛋白，其运输控制着其在细胞表面的可用性，从而在生理和病理条件下塑造了谷氨酸能神经传递。已知细胞外谷氨酸可触发从细胞表面到细胞内区室的泛素依赖性GLT-1内在化，但在这里我们显示内在化还需要钙离子的参与。与以前的研究一致，向混合的原代培养物（含有神经元和星形胶质细胞）中添加谷氨酸（1 mM）会促进GLT-1的内在化，这种作用在没有细胞外Ca ²⁺的情况下被抑制。Ca ²⁺的途径使用遗传编码的钙传感器GCaMP6f在这些混合培养物中分析了星形胶质细胞的动员。谷氨酸激活复杂的钙进入模式，尤其是在暴露于谷氨酸之后的初始阶段，谷氨酸转运蛋白部分驱动细胞内Ca ²⁺浓度急剧而迅速地升高。Na ⁺ / Ca ²⁺交换子（NCX）在此Ca ^2+中起主导作用动员及其阻滞抑制了星形胶质细胞和更直接的实验系统（例如用GLT-1瞬时转染的HEK293细胞）中谷氨酸诱导的GLT-1的内在化。在局部缺血或脑外伤等情况下，这种调节机制可能与控制细胞表面GLT-1转运蛋白的量有关，在这种情况下，细胞外谷氨酸的浓度持续升高，并促进Ca ^2+的快速动员。