Myelin basic protein as a binding partner and calmodulin adaptor for the BKCa channel.
The activity and localization of large-conductance Ca2+ -activated K+ (BKCa) channels are known to be modulated by several different proteins. Although many binding partners have been identified via yeast two-hybrid screening, this method may not detect certain classes of interacting proteins such as low affinity binding proteins or multi-component protein complexes. In this study, we employed mass spectrometry to identify proteins that interact with BKCa channels. We expressed and purified the 'tail domain' of the rat BKCa channel alpha-subunit, a 54-kDa region that is crucial for expression and functional activity of the channel. Using rat brain lysate and purified 'tail domain', we identified several novel proteins that interact with the BKCa channel. These included the myelin basic protein (MBP), upon which we performed subsequent biochemical and electrophysiological studies. Interaction between the BKCa channel and MBP was confirmed in vivo and in vitro. MBP co-expression affected the Ca2+ -dependent activation of the BKCa channel by increasing its Ca2+ sensitivity. Moreover, we showed that calmodulin (CaM) interacts with the BKCa channel via MBP. Since CaM is a key regulator of many Ca2+ -dependent processes, it may be recruited by MBP to the vicinity of the BKCa channel, modulating its functional activity.
Kim
Hyunyoung
H
Department Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea.
Jo
Sooyeon
S
Song
Hye-Jin
HJ
Park
Zee-Yong
ZY
Park
Chul-Seung
CS
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002540:Cerebral Cortex / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016622:Silver Staining; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; C436006:Mbp protein, rat; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D024681:Potassium Channels, Calcium-Activated; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase