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

Abstract

The CorA Mg(2+) channel is the primary uptake system in about half of all bacteria and archaea. However, the basis for its Mg(2+) selectivity is unknown. Previous data suggested that CorA binds a fully hydrated Mg(2+) ion, unlike other ion channels. The crystal structure of Thermotoga maritima CorA shows a homopentamer with two transmembrane segments per monomer connected by a short periplasmic loop. This highly conserved loop, (281)EFMPELKWS(289) in Salmonella enterica serovar Typhimurium CorA, is the only portion of the channel outside of the cell, suggesting a role in cation selectivity. Mutation of charged residues in the loop, E281 and K287, to any of several amino acids had little effect, demonstrating that despite conservation electrostatic interactions with these residues are not essential. While mutation of the universally conserved E285 gave a minimally functional channel, E285A and E285K mutants were the most functional, again indicating that the negative charge at this position is not a determining factor. Several mutations at K287 and W288 behaved anomalously in a transport assay. Analysis indicated that mutation of K287 and W288 disrupts cooperative interactions between distinct Mg(2+) binding sites. Overall, these results are not compatible with electrostatic interaction of the Mg(2+) ion with the periplasmic loop. Instead, the loop appears to form an initial binding site for hydrated Mg(2+), not for the dehydrated cation. The loop residues may function to accelerate dehydration of the before entry of Mg(2+) into the pore of the channel.

Figure 1. Water exchange rates for selected cations

Adapted from a similar figure in Hille (27) with additional data (56). The selective inhibitor of CorA, CoHex, is also shown; however, the exchange rate for CoHex is for the ammine groups, not water.

Figure 2. Properties of the periplasmic loop of the CorA Mg²⁺ channel

Conservation of amino acids at specific positions within the periplasmic loop is shown, based on the loop sequences shown in Supplemental Figure 1.

Figure 3. ⁶³Ni²⁺ uptake of E281 mutants

A. Mg²⁺ dose response curves for E281K, E281R, E281D single mutants and the E281K/K287E double mutant. B. Mg²⁺ dose response curves for E281A, E281L, E281N and E281Q single mutants. Each curve is normalized to maximal uptake for that strain in the absence of competing cation. The data are an average of at least 4 independent experiments done in triplicate. In a typical experiment, uptake ranged from 10,000 cpm for wild type to 1,500 cpm for E281N, over a background (10 mM Mg²⁺) of ~600 cpm. C. Maximal ⁶³Ni²⁺ uptake determined by incubating cells for 10 min with ~0.3 μCi ⁶³Ni²⁺ per tube with a total [Ni²⁺] of 125 μM. Uptake in the presence of 10 mM Mg²⁺ (~600 cpm) was taken as background and subtracted from total uptake to give net cpm uptake. The data are representative of at least three independent experiments done in triplicate.

Figure 4. ⁶³Ni²⁺ uptake of K287 mutants

Dose response curves for K287D, K287A, K287E, K287R and K287Q mutants. Each curve is normalized to uptake for that strain in the absence of competing cation. A. Mg²⁺ dose response curve. B. Ni²⁺ dose response curve. C. Co²⁺ dose response curve. D. CoHex dose response curve. Data are representative of at least three independent experiments done in triplicate. In a typical experiment, uptake ranged from 30,000 cpm for wild type to 10,000 cpm for K287D, over a background (10 mM Mg²⁺) of ~600 cpm. Error bars are not shown for clarity but were similar to those shown in Figure 3.

Figure 5. Analysis of E285 mutants

A. Maximal ⁶³Ni²⁺ uptake. Cells were incubated for 30 min with 10μCi ⁶³Ni²⁺ per tube with a total [Ni²⁺] of 125 μM. Uptake in the presence of 50 mM Mg²⁺ (~1500 cpm) was taken as background and subtracted from total uptake to give net cpm uptake. The data are representative of at least two independent experiments with each mutant done in triplicate. B. Growth complementation of MM281 by E285 mutants. As described in Methods, OD_600nm was measured every 20 min for 14 hr. The data presented are at 6 hr growth and are representative of three independent experiments done in triplicate. For clarity, error bars are drawn in one direction only.

Figure 6. ⁶³Ni²⁺ uptake of F282 mutants

The Mg²⁺ dose response curves for F282A, F282C, F282W and F282Y single mutants shown. Each curve is normalized to uptake for that mutant in the absence of competing cation. In a typical experiment, uptake ranged from 40,000 cpm for wild type to 25,000 cpm for F282C, over a background (10 mM Mg²⁺) of 600 cpm. Inset: To determine maximal ⁶³Ni²⁺ uptake, cells were incubated for 10 min with ~0.3 μCi ⁶³Ni²⁺ per tube with total [Ni²⁺] of 125μM. Uptake in the presence of 10 mM Mg²⁺ (~600 cpm) was taken as background and subtracted from total uptake to give net cpm uptake. The data are representative of at least two independent experiments done in triplicate. Error bars are not shown for clarity but were similar to those shown in Figure 3.

Figure 7. ⁶³Ni²⁺ uptake of W288 mutants

A. To determine maximal ⁶³Ni²⁺ uptake, cells were incubated for 10 min with 0.3 μCi ⁶³Ni²⁺ per tube with a total [Ni²⁺] of 125μM. Uptake in the presence of 10 mM Mg²⁺ (~600 cpm) was taken as background and subtracted from total uptake to give net cpm uptake. The data are representative of at least two independent experiments done in triplicate. B. Mg²⁺ dose response curve. C. Co²⁺ dose response curve. D. Ni²⁺ dose response curve. Each curve is normalized to uptake for that mutant in the absence of competing cation. Data are representative of at least three independent experiments done in triplicate. Error bars are not shown for clarity but were similar to those shown in Figure 3. Maximal uptake for these mutants ranged from ~3,000 to 8,000 cpm.

Figure 8. ⁶³Ni²⁺ uptake of the K287D mutant

The ability of Mg²⁺ to inhibit ⁶³Ni²⁺ uptake was measured at different total Ni²⁺ concentrations. Each curve is normalized to uptake in the absence of competing cation at the indicated Ni²⁺ concentration. Data are representative of at least two independent experiments done in triplicate. Error bars are not shown for clarity but were similar to those shown in Figure 3. Maximal uptake for these mutants ranged from ~2,000 to 30,000 cpm.