Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening.

The CorA Mg2+ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg2+ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg2+ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg2+ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3 Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg2+ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of 63Ni2+, indicating functionality. Thus, absence of Mg2+ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg2+ channel and not a Co2+ channel.