Differential roles of blocking ions in KirBac1.1 tetramer stability

Abstract

Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.

FIGURE 1.

Purification and tetramer stability of WT KirBac1.1 and mutants A109C and I131C. A, representative SDS-PAGE of purified WT KirBac1.1, A109C, and I131C proteins. All experiments in this figure and the following figures were performed at an ambient temperature of 22 °C. Bands consistent with the dimer are present at ∼70–80 kDa for A109C and I131C. A high molecular mass band at ∼160 kDa, consistent with the tetramer, is seen for only I131C. Tetramer (T), dimer (D), and monomer (M) are shown to the right of the gel. B, ribbon diagram of the KirBac1.1 crystal structure showing only the distal portion of the pore helix, the selectivity filter, and the second transmembrane α-helix. Ala-109 and Ile-131 are highlighted. C, size exclusion chromatography of purified WT KirBac1.1, A109C, and I131C using a Superdex 200 column. The running buffer contained 50 mm Tris-HCl, pH 8.0, 150 mm KCl, and 1.0 mm tridecylmaltoside. The position of KcsA, presumed tetrameric, is marked. mAU, milliabsorbance units.

FIGURE 2.

Barium stabilization of the KirBac1.1 tetramer. A, representative SDS-polyacrylamide gels of purified WT KirBac1.1, A109C, or I131C in the presence of varying concentrations of BaCl₂. Purified KirBac1.1 proteins were preincubated with BaCl₂ for 10 min, mixed with 5× protein loading buffer without dithiothreitol, and loaded onto a 4–15% gradient gel. T, tetramer; M, monomer. B, tetramer formation as a function of [Ba²⁺]. The relative density of the tetramer band was estimated by densitometry. Each data point represents an average of three separate gels. C, barium block of KirBac1.1 channel activity. Purified KirBac1.1 channels were reconstituted into liposomes (1-palmitoyl-2-oleoylphosphatidylethanolamine/1-palmitoyl-2-oleoylphosphatidylglycerol at a ratio of 9:1), and ⁸⁶Rb⁺ uptake was measured at 45 s in the presence of varying concentrations of external BaCl₂ (n = 3–5 for each point). Uptake was normalized to maximal uptake as assessed by valinomycin.

FIGURE 3.

Magnesium blocks KirBac1.1 but does not stabilize the tetramer. A, shown is a representative SDS-polyacrylamide gel of WT KirBac1.1 protein preincubated with MgCl₂ for 10 min, mixed with 5× protein loading buffer without dithiothreitol, and loaded onto a 4–15% gradient gel. M, monomer. B, tetramer stabilization by Ba²⁺ is not sensitive to the presence of Mg²⁺. WT KirBac1.1 protein was preincubated in 10 mm MgCl₂ and varying [BaCl₂] for 10 min prior to loading onto SDS-polyacrylamide gel. Data points represent fractional tetramer formation as a function of [Ba²⁺]. C, purified KirBac1.1 channels were reconstituted into liposomes (POPE/POPG at a ratio of 9:1), and ⁸⁶Rb⁺ uptake was measured at 45 s in the presence of varying concentrations of external MgCl₂ (n = 3 for each point and normalized to maximal uptake as assessed by valinomycin). The smooth line is a fitted Hill equation (see “Materials and Methods”) with H = 0.98 and K_1/2 = 78 μm.

FIGURE 4.

Cd²⁺ stabilizes the KirBac1.1(I138C) tetramer. A, SDS-PAGE of WT KirBac1.1 protein (left lane) or WT KirBac1.1 preincubated in 10 mm Cd²⁺ prior to loading onto the gel (right lane). B, representative SDS-PAGE of KirBac1.1(I138C). Mutant protein was preincubated with varying [Cd²⁺] for 10 min prior to loading onto the gel. Tetramer (T), dimer (D), and monomer (M) are shown to the right of the gel. C, [Cd²⁺] dependence of tetramer formation. Data points represent average densitometry measurements of the tetramer band from gels as in B (n = 3 for each point). The curve represents a fitted Hill equation (see “Materials and Methods”) with H = 1.7 and K_1/2 = 687 μm. D, ribbon diagram of KirBac1.1, with Ile-138 highlighted.

FIGURE 5.

Cd²⁺ block of cysteine-substituted KirBac1.1 mutants. A, ⁸⁶Rb⁺ uptake into liposomes reconstituted with KirBac1.1 cysteine mutants was measured at 60 s at varying external Cd²⁺ concentrations. Bars represent the [Cd²⁺] needed to block 50% of maximal uptake (K_1/2) for each mutant (n = 3–7). Mutants labeled with asterisks were nonfunctional. NE, no bacterial expression of protein. Inset, Fourier transform power spectrum of the K_1/2 data (see “Materials and Methods”). The major peak occurs at 92° with an α-periodicity index of 3.2, consistent with the α-helical structure of TM2. The peak of an ideal α-helix is marked by an arrow. B, a helical wheel plot of residues in the second transmembrane α-helix of KirBac1.1 is shown. Cysteine substitution of residues that increased or decreased sensitivity to Cd²⁺ block as compared with WT is colored red or blue, respectively. Cysteine substitution of residues that resulted in nonfunctional channels or no expression are in white. C, pore-facing cysteine-substituted residues confer increased sensitivity to Cd²⁺ block. The space-filled structure of the KirBac1.1 channel is shown, with one monomer moved to the left to show the central pore. The monomer on the left has been rotated 180° to show pore-facing residues. The cytoplasmic domains have been removed for clarity. In each view, the arrow represents the central axis of the channel for reference. Residues that confer increased sensitivity to Cd²⁺ block are colored red.

FIGURE 6.

Competitive and additive effects of Cd²⁺ and Ba²⁺ on WT KirBac1.1 and I138C tetramer stability. A and C, representative SDS-PAGE of WT KirBac1.1 and I138C proteins, respectively, preincubated with Cd²⁺ and Ba²⁺ at the concentrations indicated prior to loading onto the gel. B and D, Ba²⁺ and Cd²⁺ dependence of tetramer formation. Data points represent average densitometry measurements of the tetramer band from gels as in A and C for WT KirBac1.1 (B) or I138C (D) protein (n = 3 for each point).