Luo Xiang, Ahn Wooin, Muallem Shmuel, Zeng Weizhong
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75390-9040, USA.
Methods Enzymol. 2004;389:119-30. doi: 10.1016/S0076-6879(04)89008-6.
Analysis of the function of regulator of G-protein signaling (RGS) protein function and their selectivity of action in vivo is complicated by the expression of multiple RGS proteins in a single cell and requires precise control of cytosolic RGS protein concentration. This article describes two experimental systems using pancreatic acinar cells suitable for such analyses. The first is pancreatic acini permeabilized with streptolysin O, which retains agonist responsiveness while allowing RGS proteins and molecules with molecular masses of up to 25-30 kDa access to the cytosol. The second is a whole cell recording of the Ca(2+)-activated Cl- current of single pancreatic acinar cells as a reporter of [Ca2+]i. This system can be used to introduce to the cytosol any protein of interest, including recombinant RGS proteins and RGS protein-scavenging antibodies. The use of these systems to study the specificity of RGS proteins action, the function of their domains, and the role of RGS proteins in controlling Ca2+ oscillations is discussed.
由于单个细胞中多种G蛋白信号调节蛋白（RGS）的表达，对其在体内的功能及其作用选择性进行分析变得复杂，这需要精确控制胞质RGS蛋白浓度。本文描述了两种适用于此类分析的利用胰腺腺泡细胞的实验系统。第一种是用链球菌溶血素O通透处理的胰腺腺泡，它保留了激动剂反应性，同时允许RGS蛋白和分子量高达25 - 30 kDa的分子进入胞质溶胶。第二种是对单个胰腺腺泡细胞的Ca(2 +)激活的Cl - 电流进行全细胞记录，以此作为[Ca2 +]i的报告指标。该系统可用于将任何感兴趣的蛋白引入胞质溶胶，包括重组RGS蛋白和RGS蛋白清除抗体。讨论了使用这些系统研究RGS蛋白作用的特异性、其结构域的功能以及RGS蛋白在控制Ca2 +振荡中的作用。
