A MITOCHONDRIAL DOOR FOR CALCIUM Free

Calcium (Ca²⁺) signalling is one of the main mechanisms that cells use to integrate and process intra- and extracellular signals. Signal transduction occurs when the cell is stimulated to release Ca²⁺ from intracellular stores. The resulting increase in the cytosolic Ca²⁺ concentration is sensed by various proteins and converted into an appropriate cellular response. For this system to function, cells must maintain a low cytosolic Ca²⁺ concentration by continuously extruding Ca²⁺ ions from the cytosol into intracellular stores. The mitochondrion is an important Ca²⁺ store that takes up, but also releases, Ca²⁺ ions on demand. Although the proteins that move Ca²⁺ in and out of mitochondria have been studied for decades, the molecular identity of these proteins remained unknown until David Clapham and colleagues from the Harvard Medical School identified one of the elusive calcium ion transport proteins in a recent Science paper.

Knowing that calcium must be transported by proteins such as calcium channels, or Ca²⁺/Na⁺ and Ca²⁺/H⁺ exchangers, across the inner mitochondrial membrane, the team decided to try to identify the genes that encode proteins involved in mitochondrial Ca²⁺ transport. They designed a clever high-throughput assay to detect calcium transporters based on a Ca²⁺ and H⁺ (pH) sensing fluorescent protein. Generating Drosophila cells that were equipped with this fluorescent protein, the team determined the concentrations of Ca²⁺ and H⁺ ions in the cells' mitochondria by measuring the amount of florescence at different excitation wavelengths. Then they treated these cells with double stranded RNA molecules (dsRNA) to each of the ~20,000 Drosophila genes in the hope of inactivating (knocking down) the expression of a gene involved in the mitochondrial Ca²⁺ transport.

The team identified one gene whose knockdown abolished mitochondrial Ca²⁺ and pH increases: a homologue of human Letm1, an evolutionarily conserved protein of unknown function. In a series of subsequent experiments performed in Drosophila and human cells, the team discovered that Letm1 functions in the slow uptake of Ca²⁺ into mitochondria in exchange for H⁺ ions observed at submicromolar Ca²⁺ concentrations.

To test more directly whether Letm1 is a Ca²⁺/H⁺ exchanger, the team expressed Letm1 in bacteria, purified the protein and integrated it into the membranes of liposomes, tiny membrane-enclosed bubbles filled with and surrounded by defined buffers. Then they measured Ca²⁺ uptake and pH changes in the liposomes using fluorescent dyes. They showed that the Letm1 protein is responsible for the specific uptake of Ca²⁺ by liposomes, and that the Ca²⁺ transport was dependent on H⁺ ions. Moreover, the exchanger turned out to be electrogenic, because a twofold positively charged Ca²⁺ ion is exchanged for a single positively charged H⁺ ion resulting in the generation of a potential difference across the membrane.

By identifying the mitochondrial Ca²⁺/H⁺ exchanger, Letm1, the Harvard team has supplied an important piece to the puzzle of mitochondrial Ca²⁺ transport. The reported transport properties of Lemt1 indicate that Ca²⁺ entry is mediated by Lemt1 but is limited by the mitochondrial pH gradient. As Letm1 is genetically linked to Wolf—Hirschhorn syndrome, a genetic disorder characterized by mental retardation, microcephaly, seizures and hypotonia, its discovery may also help to understand the underlying pathophysiology of this devastating disease.