The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel.
The epithelial Ca(2+) channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca(2+) (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 Ca(2+) permeation of ECaC and modulation by extracellular Mg(2+). Mutation of the aspartate residue, D542A, abolishes Ca(2+) permeation and Ca(2+)-dependent current decay as well as block by extracellular Mg(2+), whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca(2+) permeability and Ca(2+)-dependent current decay. Block of monovalent currents through ECaC by Mg(2+) 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. Glu(535) and Asp(550), had only minor effects on Ca(2+) permeation properties.
Nilius
B
B
Department of Physiology, Campus Gasthuisberg, KU Leuven, Leuven B-3000, Belgium. bernd.nilius@med.kuleuven.ac.be
Vennekens
R
R
Prenen
J
J
Hoenderop
J G
JG
Droogmans
G
G
Bindels
R J
RJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050916:TRPV Cation Channels; D014162:Transfection
D015220:Calcium Channels; C118497:ECaC protein, Oryctolagus cuniculus; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C492240:Trpv5 protein, mouse; D001224:Aspartic Acid; D008274:Magnesium; D002118:Calcium