Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1

J Gen Physiol. 2014 Jan;143(1):67-73. doi: 10.1085/jgp.201311096. Epub 2013 Dec 16.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiporters / metabolism*
  • Calcium / metabolism
  • Calcium-Binding Proteins / metabolism*
  • Cation Transport Proteins / metabolism*
  • Cations, Divalent / metabolism
  • Clonazepam / analogs & derivatives
  • Clonazepam / pharmacology
  • Humans
  • Ion Transport
  • Liposomes / metabolism
  • Membrane Proteins / metabolism*
  • Mitochondrial Proteins / metabolism*
  • Potassium / metabolism
  • Ruthenium Red / pharmacology
  • Sodium / metabolism
  • Thiazepines / pharmacology

Substances

  • Antiporters
  • Calcium-Binding Proteins
  • Cation Transport Proteins
  • Cations, Divalent
  • LETM1 protein, human
  • Liposomes
  • Membrane Proteins
  • Mitochondrial Proteins
  • Thiazepines
  • calcium-hydrogen antiporters
  • Ruthenium Red
  • Clonazepam
  • CGP 37157
  • Sodium
  • Potassium
  • Calcium