Warsi Jamshed, Luo Dong, Elvira Bernat, Jilani Kashif, Shumilina Ekaterina, Hosseinzadeh Zohreh, Lang Florian
Department of Physiology I, University of Tuebingen, Gmelinstr. 5, 72076, Tuebingen, Germany.
J Membr Biol. 2014 Aug;247(8):713-20. doi: 10.1007/s00232-014-9695-3. Epub 2014 Jun 14.
Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3(A572V) is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3(K855A). Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3(A568V) or inactive JAK3(K851A), and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 (jak3(-/-)) and from corresponding wild-type mice (jak3(+/+)). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current (Ig), which was significantly increased following coexpression of JAK3. Ig in oocytes expressing EAAT3 was further increased by JAK3(A568V) but not by JAK3(K851A). Ig in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal Ig and significantly reduced the glutamate concentration required for half maximal Ig (Km). Intestinal electrogenic glutamate transport was significantly lower in jak3(-/-) than in jak3(+/+) mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms.
Janus激酶3（JAK3）参与细胞因子受体信号传导，赋予细胞存活能力并刺激细胞增殖。功能获得性突变JAK3（A572V）见于急性巨核细胞白血病。用丙氨酸取代ATP配位赖氨酸会产生无活性的JAK3（K855A）。最近的观察结果揭示了JAK3调节离子转运的能力。因此，本研究探讨了JAK3是否调节谷氨酸转运体EAAT1 - 4，这些载体在包括肠细胞、肾细胞、神经胶质细胞和神经元在内的多种细胞中完成谷氨酸和天冬氨酸的转运。为此，将EAAT1、2、3或4在非洲爪蟾卵母细胞中表达，同时有或没有额外表达小鼠野生型JAK3、组成型活性JAK3（A568V）或无活性JAK3（K851A），并通过双电极电压钳测定电生性谷氨酸转运。此外，采用尤斯灌流小室测定缺乏功能性JAK3的小鼠（jak3（-/-））和相应野生型小鼠（jak3（+/+））肠道中的电生性谷氨酸转运。结果，在表达EAAT1、2、3或4的卵母细胞中，但在注射水的卵母细胞中未出现，向细胞外浴中添加谷氨酸会产生内向电流（Ig），在共表达JAK3后该电流显著增加。表达EAAT3的卵母细胞中的Ig在JAK3（A568V）存在时进一步增加，但在JAK3（K851A）存在时未增加。在表达EAAT3 + JAK3的卵母细胞中，JAK3抑制剂WHI - P154（22 μM）可使Ig显著降低。动力学分析表明，JAK3增加了最大Ig，并显著降低了达到最大Ig一半时所需的谷氨酸浓度（Km）。jak3（-/-）小鼠肠道中的电生性谷氨酸转运明显低于jak3（+/+）小鼠。总之，JAK3是兴奋性氨基酸转运体亚型的强大调节因子。
