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 Ca2+ and K+ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca2+ fluorophore and 45Ca2+-based assays, we demonstrate directly that Letm1 is a Ca2+ transporter, with apparent affinities of cations in the sequence of Ca2+ ≈ Mn2+ > Gd3+ ≈ La3+ > Sr2+ >> Ba2+, Mg2+, K+, Na+. Kinetic analysis yields a Letm1 turnover rate of 2 Ca2+/s and a Km of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca2+/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+/Ca2+ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H+-dependent Ca2+ transport mechanism identified in intact mitochondria.