Analyses of RGS protein control of agonist-evoked Ca2+ signaling.
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.
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75390-9040, USA.
Xiang
X
Luo
Wooin
W
Ahn
Shmuel
S
Muallem
Weizhong
W
Zeng
D000814:Aniline Compounds; D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D020962:Heterotrimeric GTP-Binding Proteins / Q000819:agonists* / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013301:Streptolysins / Q000494:pharmacology; D014966:Xanthenes
D000814:Aniline Compounds; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D020710:RGS Proteins; D011994:Recombinant Proteins; D013301:Streptolysins; D014966:Xanthenes; C025964:streptolysin O; C059715:Fluo-3; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium