The Single Pore Residue Asp542 Determines Ca2+ Permeation and Mg2+ Block of the Epithelial Ca2+ Channel

The epithelial Ca2+ channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca2+ (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region be...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-01, Vol.276 (2), p.1020-1025
Main Authors: Nilius, Bernd, Vennekens, Rudi, Prenen, Jean, Hoenderop, Joost G.J., Droogmans, Guy, Bindels, Rene J.M.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Animals
Aspartic Acid
Calcium - metabolism
Calcium Channels - chemistry
Calcium Channels - drug effects
Calcium Channels - physiology
Humans
Kidney - metabolism
Kinetics
Magnesium - pharmacology
Membrane Potentials - drug effects
Membrane Potentials - physiology
Mice
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Structure, Secondary
Rabbits
Recombinant Proteins - chemistry
Recombinant Proteins - drug effects
Recombinant Proteins - metabolism
Sequence Alignment
Sequence Homology, Amino Acid
Transfection
TRPV Cation Channels
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Summary:The epithelial Ca2+ channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca2+ (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca2+ permeation of ECaC and modulation by extracellular Mg2+. Mutation of the aspartate residue, D542A, abolishes Ca2+ permeation and Ca2+-dependent current decay as well as block by extracellular Mg2+, whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca2+ permeability and Ca2+-dependent current decay. Block of monovalent currents through ECaC by Mg2+ was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e.Glu535 and Asp550, had only minor effects on Ca2+ permeation properties.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M006184200