QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:
Institution: Universidad Austral de Chile-Biblioteca Central Sign In as Member / Individual

Full Text of this Article Reprint (PDF) Version of this Article Similar articles found in: PNAS Online ISI Web of Science PubMed PubMed Citation This Article has been cited by: other online articles Search PubMed for articles by: Bian, S. || Moczydlowski, E. Search for citing articles in: ISI Web of Science (16) Alert me when: new articles cite this article Download to Citation Manager

Physiology
Ca²⁺-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca²⁺-dependent activation

Shumin Bian^*, Isabelle Favre^*,, and Edward Moczydlowski^*,,§

Departments of ^* Pharmacology and  Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066

Communicated by Ramon Latorre, Center for Scientific Studies of Santiago, Valdivia, Chile, February 13, 2001 (received for review November 28, 2000)

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca²⁺-activated K⁺ channel (BK channel) contains Ca²⁺-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca²⁺ 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 His₆-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a ⁴⁵Ca²⁺-overlay protein blot assay to detect Ca²⁺ binding. Dslo-C280 exhibits specific binding of ⁴⁵Ca²⁺ in comparison with various control proteins and known EF-hand Ca²⁺-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the "Ca-bowl" motif are changed to Asn, reduces ⁴⁵Ca²⁺-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca²⁺ sensitivity for activation and a shift of +80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca²⁺ and a reduction in apparent Ca²⁺ affinity, suggesting the loss of one Ca²⁺-binding site per monomer. These results demonstrate a functional correlation between Ca²⁺ binding to a specific region of the BK protein and Ca²⁺-dependent activation, thus providing a biochemical approach to study this process.

www.pnas.org/cgi/doi/10.1073/pnas.081072398

This article has been cited by other articles:

				X. Qian and K. L. Magleby {beta}1 subunits facilitate gating of BK channels by acting through the Ca2+, but not the Mg2+, activating mechanisms PNAS, August 19, 2003; 100(17): 10061 - 10066. [Abstract] [Full Text] [PDF]

				R. J Lang, J. R. Harvey, and E. L Mulholland Sodium (2-sulfonatoethyl) methanethiosulfonate prevents S-nitroso-L-cysteine activation of Ca2+-activated K+ (BKCa) channels in myocytes of the guinea-pig taenia caeca Br. J. Pharmacol., July 6, 2003; 139(6): 1153 - 1163. [Abstract] [Full Text] [PDF]

				A. Anishkin, V. Gendel, N. A. Sharifi, C.-S. Chiang, L. Shirinian, H. R. Guy, and S. Sukharev On the Conformation of the COOH-terminal Domain of the Large Mechanosensitive Channel MscL J. Gen. Physiol., March 1, 2003; 121(3): 227 - 244. [Abstract] [Full Text] [PDF]

				K. L. Magleby Gating Mechanism of BK (Slo1) Channels: So Near, Yet So Far J. Gen. Physiol., February 1, 2003; 121(2): 81 - 96. [Full Text] [PDF]

				S.-X. Wang, M. Ikeda, and W. B. Guggino The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression J. Biol. Chem., January 24, 2003; 278(4): 2713 - 2722. [Abstract] [Full Text] [PDF]

				X. Qian, C. M. Nimigean, X. Niu, B. L. Moss, and K. L. Magleby Slo1 Tail Domains, but Not the Ca2+ Bowl, Are Required for the {beta}1 Subunit to Increase the Apparent Ca2+ Sensitivity of BK Channels J. Gen. Physiol., December 1, 2002; 120(6): 829 - 843. [Abstract] [Full Text] [PDF]

				X. Niu and K. L. Magleby Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca2+ PNAS, August 20, 2002; 99(17): 11441 - 11446. [Abstract] [Full Text] [PDF]

				L. Bao, A. M. Rapin, E. C. Holmstrand, and D. H. Cox Elimination of the BKCa Channel's High-Affinity Ca2+ Sensitivity J. Gen. Physiol., July 30, 2002; 120(2): 173 - 189. [Abstract] [Full Text] [PDF]

				P. Orio, P. Rojas, G. Ferreira, and R. Latorre New Disguises for an Old Channel: MaxiK Channel {beta}-Subunits News Physiol Sci, August 1, 2002; 17(4): 156 - 161. [Abstract] [Full Text] [PDF]

				N. Mallouk and B. Allard Ca2+ Influx and Opening of Ca2+-Activated K+ Channels in Muscle Fibers from Control and mdx Mice Biophys. J., June 1, 2002; 82(6): 3012 - 3021. [Abstract] [Full Text] [PDF]

				B. L. Moss and K. L. Magleby Gating and Conductance Properties of BK Channels Are Modulated by the S9-S10 Tail Domain of the {alpha} Subunit: A Study of mSlo1 and mSlo3 Wild-type and Chimeric Channels J. Gen. Physiol., November 26, 2001; 118(6): 711 - 734. [Abstract] [Full Text] [PDF]