膜片钳全细胞记录法在研究谷氨酰胺转运蛋白分子机制中的应用

Application of patch clamp whole-cell recording method to study on molecular mechanism of glutamine transporters

为了研究中性氨基酸转运蛋白-谷氨酰胺转运蛋白的结构与功能,采用膜片钳全细胞记录法测定谷氨酰胺转运蛋白2介导的电流.结果表明:谷氨酰胺转运蛋白诱导了底物氨基酸和Na+浓度依赖性电流,可用于测定底物氨基酸和Na+对转运蛋白的表观亲和常数Km,SNAT2野生型对丙氨酸的表观亲和常数KmA la为(200±5)×10-6mol/L,对Na+的表观亲和常数KmNa为(100±7)×10-3mol/L.谷氨酸转运蛋白转运底物氨基酸过程是生电的过程,在膜电势负值时氨基酸转运电流增大,可用于转运机制的研究.因此膜片钳全细胞记录法是研究谷氨酰胺转运蛋白分子机制的有效方法.

Patch clamp whole-cell recording method was used in order to study on structure and function of neutral amino acid transporters-glutamlne transporters. Our results showed that glutamine transporter 2 induced transport currents dependent on amino acid and anion leak currents dependent on extracellular Na^＋. The apparent affinity was （200±5） × 10^-6mol/L for SNAT2 to alanine and （100±7） × 10^-3mol/L for SNAT2 to Na^＋ , respectively. The process of SNAT2 transport amino acid was electrogenic. The transport currents were increased with electronic driving force increasing. In conclusion, patch clamp whole-cell recording method is a valid technique for study on molecular mechanism of glutamine transporters.