Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

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 (∼40 kDa) on SDS-PAGE. Addition of Ba²⁺ (K_1/2 ∼ 50 μm) prior to loading results in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue located near the expected Ba²⁺-binding site, decreased tetramer stabilization by Ba²⁺ (K_1/2 ∼ 300 μm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba²⁺. Neither mutation affected Ba²⁺ block of channel activity (using ⁸⁶Rb⁺ flux assay). In contrast to Ba²⁺, Mg²⁺ had no effect on tetramer stability (even though Mg²⁺ was a potent blocker). Many studies have shown Cd²⁺ 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 Cd²⁺ on KirBac1.1 with cysteine substitutions in M2. Cd²⁺ block potency followed an α-helical pattern consistent with the crystal structure. Significantly, Cd²⁺ strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba²⁺, consistent with both ions simultaneously occupying the channel: Ba²⁺ at the selectivity filter entrance and Cd²⁺ coordinated by I138C side chains in the inner cavity.Potassium channels are expressed in many cell types and are key players in a wide range of physiological processes. One subset of potassium channels, the inward-rectifying potassium (Kir) channels, are functionally blocked by cytosolic cations such as Mg²⁺ and polyamines and contribute to the regulation of membrane excitability, cardiac rhythm, vascular tone, insulin release, and salt flow across epithelia (1–3). There are seven subfamilies of eukaryotic Kir channel genes. Among them, Kir1 encodes weak rectifiers, whereas Kir2 and Kir5 encode strong rectifiers; Kir3 encodes G-protein-regulated channels; and Kir6 encodes ATP-sensitive channels (4). Recently, a related bacterial family of genes (KirBac) has been identified (5, 6), and in 2003, the first member (KirBac1.1) was crystallized (7), providing a structural model for eukaryotic channels.The crystal structure of KirBac1.1 revealed a tetrameric pore structure similar to that seen in KcsA and a novel cytoplasmic domain (7, 8). The selectivity filter of both KirBac1.1 and KcsA consists of an extremely conserved pore loop followed by a central cavity, forming a transmembrane ion-selective permeation pore (7, 8). The linear arrangement of five oxygen rings (four from carbonyl oxygens and one from a Thr side chain) in the selectivity filter coordinates with ions, compensating for the energy barrier caused by K⁺ dehydration, thereby facilitating the rapid diffusion of K⁺ across the membrane (8–12). Two-thirds of the KirBac1.1 amino acid residues constitute the cytosolic domain that is highly conserved among the Kir subfamilies and form the cytosolic vestibule (13–16), which, together with the transmembrane pore, generates an 88-Å-long ion conduction pore (7).The prototypic potassium channel KcsA exists very stably as a tetramer, even in the harsh conditions of SDS-PAGE (17). In addition to protein-protein interaction between monomers, protein-lipid and protein-ion interactions play important roles in stabilizing the KcsA tetramer (17–20). The selectivity filter of KcsA, coordinated with K⁺ ions, can serve as a bridge between the four monomers to maintain the structure of the selectivity filter and the tetrameric architecture of the channel as a whole (11, 21). Blocking ions, such as Ba²⁺, also act as strong stabilizers (17). In the crystal structure of KcsA, Ba²⁺ occupies a site equivalent to the S4 K⁺-binding site within the selectivity filter (22). Other permeant ions (Rb⁺, Cs⁺, Tl⁺, and NH⁺₄) and strong blockers (Sr²⁺) can also contribute to the thermostability of the KcsA tetramer in SDS-PAGE (17). In contrast, impermeant ions such as Na⁺ and Li⁺ or weak blockers such as Mg²⁺ tend to destabilize the KcsA tetramer (17, 19).Like KcsA, KirBac1.1 purified using decylmaltoside or tridecylmaltoside is active and presumably stable as a tetramer in mild detergent solutions. However, in SDS-PAGE, KirBac1.1 migrates exclusively as a monomer (23). Because KcsA and KirBac1.1 are structurally similar in the transmembrane region of the pore, we hypothesized that permeant and blocking ions would also affect KirBac1.1 tetramer stability in SDS-PAGE. In the present work, the effects of blocking ions such as Ba²⁺ and Mg²⁺ on KirBac1.1 tetramer stability were examined to provide insight to the physical nature of their interaction with KirBac1.1, particularly in the selectivity filter and TM2 cavity. The data reveal important differences in the nature of the interaction of Mg²⁺ and Ba²⁺ with the channel as well as provide previously unavailable evidence for the nature of Cd²⁺ coordination within the channel.