title: Role of aspartate residues in Ca(2+) affinity and permeation of the distal ECaC1.
abstract: The Ca(2+) affinity and permeation of the epithelial Ca(2+) channel (ECaC1) were investigated after expression in Xenopus oocytes. ECaC1 displayed anomalous mole-fraction effects. Extracellular Ca(2+) and Mg(2+) reversibly inhibited ECaC1 whole cell Li(+) currents: IC(50) = 2.2 +/- 0.4 microM (n = 9) and 235 +/- 35 microM (n = 10), respectively. These values compare well with the Ca(2+) affinity of the L-type voltage-gated Ca(2+) (Ca(V)1.2) channel measured under the same conditions, suggesting that high-affinity Ca(2+) binding is a well-conserved feature of epithelial and voltage-gated Ca(2+) channels. Neutralization of D550 and E535 in the pore region had no significant effect on Ca(2+) and Mg(2+) affinities. In contrast, neutralization of D542 significantly decreased Ca(2+) affinity (IC(50) = 1.1 +/- 0.2 mM, n = 6) and Mg(2+) affinity (IC(50) > 25 +/- 3 mM, n = 4). Despite a 1,000-fold decrease in Ca(2+) affinity in D542N, Ca(2+) permeation properties and the Ca(2+)-to-Ba(2+) conductance ratio remained comparable to values for wild-type ECaC1. Together, our observations suggest that D542 plays a critical role in Ca(2+) affinity but not in Ca(2+) permeation in ECaC1.
authors.affiliation: Groupe de Recherche en Transport Membranaire, Département de Physiologie, Université de Montréal, Downtown Station, Montreal, Quebec, Canada H3C 3J7.
authors.forename: K
authors.initials: K
authors.lastname: Jean
authors.forename: G
authors.initials: G
authors.lastname: Bernatchez
authors.forename: H
authors.initials: H
authors.lastname: Klein
authors.forename: L
authors.initials: L
authors.lastname: Garneau
authors.forename: R
authors.initials: R
authors.lastname: Sauvé
authors.forename: L
authors.initials: L
authors.lastname: Parent
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004563:Electrochemistry; D005260:Female; D007686:Kidney Tubules, Distal / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D011817:Rabbits; D050916:TRPV Cation Channels; D014982:Xenopus laevis
chemical_list: D015220:Calcium Channels; C118497:ECaC protein, Oryctolagus cuniculus; D050916:TRPV Cation Channels; D001464:Barium; D001224:Aspartic Acid; D008274:Magnesium; D002118:Calcium