Mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells.
We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4)--we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.
Balana
Bartosz
B
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies. balana@salk.edu
Taylor
Natalie
N
Slesinger
Paul A
PA
D018076:DNA, Complementary / Q000235:genetics; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D018408:Patch-Clamp Techniques / Q000379:methods; D017354:Point Mutation; D043585:Receptor, Muscarinic M2 / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C484274:KCNJ5 protein, human; D043585:Receptor, Muscarinic M2