Cation selectivity by the CorA Mg2+ channel requires a fully hydrated cation.

Magnesium is an essential cation required for numerous cellular functions, including coordination to nucleotide triphosphates, membrane stability, regulation of gene transcription, DNA replication, enzyme catalysis and protein synthesis (1–5). Deficiencies in Mg2+ result in a number of pathological disease states and in turn can promote deficiencies in other important cations such as Ca2+ and K+ (6–12). The ubiquitous involvement of Mg2+ in important biological processes makes it a critical cation for study. As with any cation, transmembrane entry pathways are required. Bacterial and Archaeal species have at least three different Mg2+ uptake systems. Enterobacteria and a few other groups of bacteria express the P-type ATPases MgtA and MgtB under low Mg2+ conditions (13–15). However, the primary route for Mg2+ entry is through either the CorA or the MgtE Mg2+ channel. Either CorA or MgtE appear to be present in all fully sequenced bacterial and archaeal genomes. Approximately half of bacterial and archaeal genomes contain the CorA Mg2+ channel while the other half expresses the MgtE Mg2+ channel (16;17). A few species express both channels. The crystal structures of the Thermotoga maritima CorA (18–20) and Thermus thermophilus MgtE (21) Mg2+ channels have been recently determined. MgtE, whose eukaryotic homologs are the SLC41A family of transport systems (22), is a homodimer with a total of 10 transmembrane (TM) segments and a modular bipartite cytosolic domain (21). The CorA Mg2+ channel’s primary eukaryotic homolog is the mitochondrial Mrs2 Mg2+ channel. CorA is a homopentamer with two transmembrane segments (TM1 and TM2) per monomer. Its large N-terminal domain and the short 6 residue C-terminus are cytosolic. CorA crystal structures suggest that the pore of the protein is formed solely by TM1 (18–20). Opening of the channel appears to be controlled by Mg2+ occupation of metal binding sites in the cytosolic domain. These Mg2+ ions have been hypothesized to act as “Mg2+ sensors” such that their dissociation from CorA allows movement of the monomers, opening the channel (18–20). Molecular dynamics simulations support this interpretation (23). An even more extensive set of bound Mg2+ ions in the cytosolic domain of the MgtE Mg2+ channel appears to provide a similar function. Mutagenesis and electrophysiological analysis strongly support such a role for the bound Mg2+ in MgtE (21;24). The periplasmic end of TM1 contains the highly conserved ‘YGMNF’ motif. Mutations in this motif abolish Mg2+ transport, indicating that it is essential for function (25). Both TM segments are comprised solely of uncharged generally nonpolar amino acids. The pore appears to be lined only with backbone carbonyl groups. The few Ser and Thr residues in the membrane domain do not appear to contribute their hydroxyl groups to the pore (25). Thus, membrane flux of the most charge dense of the biological cations does not involve electrostatic interactions with the cation. This conclusion is supported by recent molecular dynamics simulations of CorA (23). The transmembrane segments are connected through the periplasm by a short, charged 9 amino acid loop. This periplasmic loop was unresolved in all reported crystal structures, most likely due to a high degree of flexibility (18–20). Because the loop is the only portion of CorA exposed on the exterior membrane face, it is logical to hypothesize that it is involved in the initial interaction of the channel with substrate cations and likely participates in cation selectivity, but the mechanism of selectivity has not been explored in any depth. Understanding the mechanism of Mg2+ selectivity with its transporters and channels necessitates consideration of the unique chemistry of the Mg2+ cation (17;26). Hydrated Mg2+ is the largest of all biological cations at 5 A in diameter, almost twice the diameter of hydrated Ca2+. The atomic ion however is among the smallest of all cations at 0.65 A. Consequently, Mg2+ undergoes a much larger change in volume from hydrated cation to atomic ion than any other cation, 400-fold versus 4–25-fold changes in volume for Na+, K+ or Ca2+. Mg2+ is invariably hexacoordinate. By contrast, Ca2+ can have several coordination states. Mg2+ holds its waters of hydration 103–104 more tightly than Ca2+, Na+ and K+. These properties result in a rigid octahedral structure in which 90 degree bond angles are strongly preferred. The rate of exchange of the waters of hydration of Mg2+ is only 105/sec, compared to ~109 for other biological cations (Figure 1) (27). This unique chemistry suggests that channels and transporters interacting with Mg2+ may have properties distinct from those of other ion transport systems (17;19;26;28). Figure 1 Water exchange rates for selected cations Recent electrophysiological experiments with the eukaryotic CorA Mg2+ channel homolog Mrs2 (29) and the bacterial MgtE Mg2+ channel (30) support this supposition. Both are ion channels with surprisingly high conductances of ~90–150 pS. Preliminary data with S. Typhimurium CorA suggests a similarly high conductance of ~140 pS (C. Obejero-Paz, A. Moomaw, S.W. Jones and M.E. Maguire, unpublished). A conductance of this magnitude indicates a flux rate >107 ions/sec through these two classes of Mg2+ channels. This rate is at least 2 orders of magnitude greater than the exchange rate of the waters of hydration surrounding Mg2+ (Figure 1) (26), an indication that the typical aqueous chemistry of Mg2+ cannot explain the measured conductance of these channels. Thus, any hypothesis concerning the mechanism of the CorA Mg2+ channel must explain not only cation selectivity but also the anomalously high conductance. Our previous work identifying cobalt(III) hexaammine (CoHex) as a selective competitive inhibitor of CorA (31) provides a starting point for investigation. Crystal structures of CoHex and similar compounds such as Ru(II) and Ru(III) hexaammine (32) show that they are comparable in size to a hydrated Mg2+ cation, ~5A. Because the ammine groups are covalently linked to Co2+ and therefore stable, these compounds represent structural analogs of the fully hydrated Mg2+ cation. Their ability to selectively inhibit CorA suggests that CorA initially interacts with a fully hydrated Mg2+ ion. The data presented in this report show that electrostatic interactions between charged residues of the periplasmic loop and the atomic (unhydrated) substrate ion do not occur. Rather, the loop (and possibly an external portion of the pore) interacts only with the fully hydrated cation. This could allow the channel to facilitate dehydration, thus allowing a partially hydrated Mg2+ to enter the pore. Thus, a significant part of the selectivity of CorA for Mg2+ derives from binding the fully hydrated cation.