Abstract

Potassium channels are tetrameric proteins that mediate K+ selective transmembrane diffusion. For the prototypic potassium channel KcsA, interactions between ions and the channel pore can modulate the stability of the tetrameric structure of KcsA, where permeant and strongly blocking ions increase stability, and impermeant or weakly blocking ions tend to decrease stability. Because of the structural similarity between the transmembrane regions of KirBac1.1 and KcsA, we examined the role of blocking ions on KirBac1.1 tetramer stability. In 150 mM KCl, purified KirBac1.1 protein migrates as a monomer (∼40kD) on SDS-PAGE. Addition of Ba2+ (K1/2 ∼ 50 μM) prior to loading results in an additional tetrameric band (∼160 kD). Mutation A109C, at a residue located near the expected Ba2+ binding site, decreased tetramer stabilization by Ba2+ (K1/2 ∼300 μM), while 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 introduced cysteines in the 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 effect of Cd2+ on KirBac1.1 with cysteine substitutions in M2. Cd2+ block potency followed an alpha-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 I38C side-chains in the inner cavity.

Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call