Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels

Wendy S VanScyoc; Rhonda A Newman; Brenda R Sorensen; Madeline A Shea

doi:10.1021/bi061134d

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Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels

Journal article

Peer reviewed

Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels

Wendy S VanScyoc, Rhonda A Newman, Brenda R Sorensen and Madeline A Shea

Biochemistry (Easton), Vol.45(48), pp.14311-14324

12/05/2006

DOI: 10.1021/bi061134d

PMID: 17128970

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Abstract

Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.

Protein Structure, Tertiary

Amino Acid Sequence

Calmodulin - genetics

Calmodulin - metabolism

Phenylalanine - metabolism

Calcium - metabolism

Paramecium - genetics

Models, Molecular

Molecular Sequence Data

Serine - genetics

Mutation - genetics

Calcium - chemistry

Serine - metabolism

Paramecium - enzymology

Animals

Ion Channels - metabolism

Phenylalanine - genetics

Nuclear Magnetic Resonance, Biomolecular

Protein Binding

Titrimetry

Calmodulin - chemistry

Binding Sites

Ion Channels - chemistry

Details

Title: Subtitle: Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels
Creators: Wendy S VanScyoc - Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109, USA
Rhonda A Newman
Brenda R Sorensen
Madeline A Shea
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.45(48), pp.14311-14324
Publisher: United States
DOI: 10.1021/bi061134d
PMID: 17128970
ISSN: 0006-2960
eISSN: 1520-4995
Grant note: R01 GM 57001 / NIGMS NIH HHS T32 GM08365-13 / NIGMS NIH HHS
Language: English
Date published: 12/05/2006
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Biochemistry and Molecular Biology
Record Identifier: 9984025298002771

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