Synthetic Peptide and Protein Domain Arrays Prepared by the SPOT Technology

The SPOT™ technology for highly parallel synthesis of peptides on flat surfaces in array type format has evolved into a versatile toolbox for a variety of applications in proteomics such as mapping protein-protein interactions and profiling the substrate specificity of enzymes such as kinases and proteases. Originally developed for the synthesis of short overlapping peptide sequences for mapping antibody epitopes this technology has recently been extended to the synthesis of functional protein domains. This opens up a variety of future applications such as target identification and protein expression profiling

Struktur des KCNJ14-Gens und funktionelle Bedeutung der Kir2.4-Kanaluntereinheit beim Menschen und bei der Maus

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Kir2.4 Surface Expression and Basal Current Are Affected by Heterotrimeric G-Proteins

Kir2.4, a strongly rectifying potassium channel that is localized to neurons and is especially abundant in retina, was fished with yeast two-hybrid screen using a constitutively active Gα(o1). Here, we wished to determine whether and how Gα(o) affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with Gα(o), and this interaction is stronger with the GDP-bound form of Gα(o). Using two-electrode voltage clamp, we recorded from oocytes that were injected with Kir2.4 mRNA and a combination of G-protein subunit mRNAs. We found that the wild type and the inactive mutant of Gα(o) reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive Gα subunits also reduce this current, whereas Gα(s) increases it. Gβγ increases the current, whereas m-phosducin, which binds Gβγ without affecting the state of Gα, reduces it. We then tested the effect of G-protein subunits on the surface expression of the channel fused to cerulean by imaging the plasma membranes of the oocytes. We found that the surface expression is affected, with effects paralleling those seen with the basal current. This suggests that the observed effects on the current are mainly indirect and are due to surface expression. Similar results were obtained in transfected HEK cells. Moreover, we show that in retinal ON bipolar cells lacking Gβ3, localization of Kir2.4 in the dendritic tips is reduced. We conclude that Gβγ targets Kir2.4 to the plasma membrane, and Gα(o) slows this down by binding Gβγ