Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing

• Published in: The Journal of general physiology 2004-05, Vol.123 (5), p.475
• Main Authors: Bao, Lin, Kaldany, Christina, Holmstrand, Ericka C, Cox, Daniel H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding sites; Calcium - metabolism; Calcium Signaling - physiology; Cells, Cultured; Cellular biology; Computer Simulation; Ion Channel Gating - physiology; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials - physiology; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oocytes - physiology; Potassium; Potassium Channels, Calcium-Activated - chemistry; Potassium Channels, Calcium-Activated - physiology; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Recombinant Proteins - metabolism; Signal Transduction - physiology; Structure-Activity Relationship; Xenopus laevis
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/jgp.200409052

There is controversy over whether Ca(2+) binds to the BK(Ca) channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca(2+) sensitivity act at the point of Ca(2+) coordination. One region in the intracellular domain that has been implicated in Ca(2+) sensing is the "Ca(2+) bowl". This region contains many acidic residues, and large Ca(2+)-bowl mutations eliminate Ca(2+) sensing through what appears to be one type of high-affinity Ca(2+)-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca(2+) bowl that are most important for Ca(2+) sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca(2+) binding to a fusion protein that contains a portion of the BK(Ca) channel's intracellular domain. Thus, much of our data supports the conclusion that Ca(2+) binds to the BK(Ca) channel's intracellular domain, and they define the Ca(2+) bowl's essential Ca(2+)-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca(2+) sensing and those that disrupt Ca(2+) binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca(2+) bowl may be in a nonnative conformation.