Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation.
Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca(2+)-activated K(+) channel (BK channel) contains Ca(2+)-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca(2+) binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His(6)-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a (45)Ca(2+)-overlay protein blot assay to detect Ca(2+) binding. Dslo-C280 exhibits specific binding of (45)Ca(2+) in comparison with various control proteins and known EF-hand Ca(2+)-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the "Ca-bowl" motif are changed to Asn, reduces (45)Ca(2+)-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca(2+) sensitivity for activation and a shift of approximately +80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca(2+) and a reduction in apparent Ca(2+) affinity, suggesting the loss of one Ca(2+)-binding site per monomer. These results demonstrate a functional correlation between Ca(2+) binding to a specific region of the BK protein and Ca(2+)-dependent activation, thus providing a biochemical approach to study this process.
Departments of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8066, USA.
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Bian
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Favre
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Moczydlowski
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004330:Drosophila; D029721:Drosophila Proteins; D006801:Humans; D015640:Ion Channel Gating; D051036:Large-Conductance Calcium-Activated Potassium Channels; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism* / Q000502:physiology; D024681:Potassium Channels, Calcium-Activated; D011485:Protein Binding
D029721:Drosophila Proteins; D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; C494844:slo protein, Drosophila; D002118:Calcium