title: Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing.
abstract: 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.
authors.affiliation: Molecular Cardiology Research Institute, New England Medical Center, Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Bao
authors.forename: Christina
authors.initials: C
authors.lastname: Kaldany
authors.forename: Ericka C
authors.initials: EC
authors.lastname: Holmstrand
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Cox
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D003198:Computer Simulation; D015640:Ion Channel Gating / Q000502:physiology; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D024681:Potassium Channels, Calcium-Activated / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D011994:Recombinant Proteins; D002118:Calcium