title: Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1.
abstract: The leucine zipper, EF hand-containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca(2+) and K(+) homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca(2+) fluorophore and (45)Ca(2+)-based assays, we demonstrate directly that Letm1 is a Ca(2+) transporter, with apparent affinities of cations in the sequence of Ca(2+) ≈ Mn(2+) > Gd(3+) ≈ La(3+) > Sr(2+) >> Ba(2+), Mg(2+), K(+), Na(+). Kinetic analysis yields a Letm1 turnover rate of 2 Ca(2+)/s and a Km of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca(2+)/2 H(+) antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H(+)-dependent Ca(2+) transport mechanism identified in intact mitochondria.
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454.
authors.forename: Ming-Feng
authors.initials: MF
authors.lastname: Tsai
authors.forename: Dawei
authors.initials: D
authors.lastname: Jiang
authors.forename: Linlin
authors.initials: L
authors.lastname: Zhao
authors.forename: David
authors.initials: D
authors.lastname: Clapham
authors.forename: Christopher
authors.initials: C
authors.lastname: Miller
mesh_terms: D017920:Antiporters / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002998:Clonazepam / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D017136:Ion Transport; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D013841:Thiazepines / Q000494:pharmacology
chemical_list: D017920:Antiporters; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D002413:Cations, Divalent; C488528:LETM1 protein, human; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D013841:Thiazepines; C027614:calcium-hydrogen antiporters; D012430:Ruthenium Red; D002998:Clonazepam; C079032:CGP 37157; D012964:Sodium; D011188:Potassium; D002118:Calcium