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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
8
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pubmed:dateCreated |
2001-4-11
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pubmed:abstractText |
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.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-10321244,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-10398695,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-10436003,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-14343300,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-1497890,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-1822543,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-1831012,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-2428908,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-2530914,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-2782390,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-6315857,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-6715311,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-7521330,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-7687074,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-7722520,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-7917297,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-8057247,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-8226763,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-8387515,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-8952957,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-8973172,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-9154910,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/11274367-9450948
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Apr
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pubmed:issn |
0027-8424
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
10
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pubmed:volume |
98
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
4776-81
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pubmed:dateRevised |
2010-9-14
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pubmed:meshHeading |
pubmed-meshheading:11274367-Animals,
pubmed-meshheading:11274367-Calcium,
pubmed-meshheading:11274367-Cell Line,
pubmed-meshheading:11274367-Drosophila,
pubmed-meshheading:11274367-Drosophila Proteins,
pubmed-meshheading:11274367-Humans,
pubmed-meshheading:11274367-Ion Channel Gating,
pubmed-meshheading:11274367-Large-Conductance Calcium-Activated Potassium Channels,
pubmed-meshheading:11274367-Patch-Clamp Techniques,
pubmed-meshheading:11274367-Potassium Channels,
pubmed-meshheading:11274367-Potassium Channels, Calcium-Activated,
pubmed-meshheading:11274367-Protein Binding
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pubmed:year |
2001
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pubmed:articleTitle |
Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation.
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pubmed:affiliation |
Departments of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8066, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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