Identification and functional characterization of ankyrin-repeat family protein ANKRA as a protein interacting with BKCa channel.
Ankyrin-repeat family A protein (ANKRA) was originally cloned in mouse as an interacting protein to megalin, a member of low-density lipoprotein receptor superfamily. Here, we report that the isolation of rat ANKRA as a new binding partner for the alpha-subunit of rat large-conductance Ca2+-activated K+ channel (rSlo). We mapped the binding region of each protein by using yeast two-hybrid and in vitro binding assays. ANKRA expressed together with rSlo channels were colocalized near the plasma membrane and coimmunoprecipitated in transfected cells. We also showed that BKCa channel in rat cerebral cortex coprecipitated with rANKRA and colocalized in cultured rat hippocampal neuron. Although the coexpression of ANKRA did not affect the surface expression of rSlo, the gating kinetics of rSlo channel was significantly altered and the effects were highly dependent on the intracellular calcium. These results indicate that ANKRA could modulate the excitability of neurons by binding directly to endogenous BKCa channel and altering its gating kinetics in a calcium-dependent manner.
Lim
Hyun-Ho
HH
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Park
Chul-Seung
CS
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism*; D019921:Biotinylation; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007700:Kinetics; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D000081033:Potassium Channels, Sodium-Activated; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques
C419548:ANKRA2 protein, human; C502820:Ankra2 protein, rat; D017487:Ankyrins; D002352:Carrier Proteins; D018076:DNA, Complementary; C494842:Kcnma1 protein, rat; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D000081033:Potassium Channels, Sodium-Activated; C000626167:kcnt1 protein, rat; D049452:Green Fluorescent Proteins; D012313:RNA; D005982:Glutathione Transferase; D002118:Calcium