Structure of the transmembrane regions of a bacterial cyclic nucleotide-regulated channel.

The six-transmembrane helix (6 TM) tetrameric cation channels form the largest ion channel family, some members of which are voltage-gated and others are not. There are no reported channel structures to match the wealth of functional data on the non-voltage-gated members. We determined the structure of the transmembrane regions of the bacterial cyclic nucleotide-regulated channel MlotiK1, a non-voltage-gated 6 TM channel.

The MlotiK1 channel transports ions along the canonical conduction pore.

Although the cyclic nucleotide-modulated potassium channel from Mesorhizobium loti, MlotiK1, is easily studied using a 86Rb+ flux assay, its comparatively low activity raises serious concerns about the integrity of the purified protein. We investigated the pathway of uptake using a multi-pronged approach. First, we probed the conduction pathway using quaternary ammonium compounds known to block conduction in eukaryotic K+ channels.

Mechanism of intracellular block of the KcsA K+ channel by tetrabutylammonium: insights from X-ray crystallography, electrophysiology and replica-exchange molecular dynamics simulations.

The mechanism of intracellular blockade of the KcsA potassium channel by tetrabutylammonium (TBA) is investigated through functional, structural and computational studies. Using planar-membrane electrophysiological recordings, we characterize the binding kinetics as well as the dependence on the transmembrane voltage and the concentration of the blocker. It is found that the apparent affinity of the complex is significantly greater than that of any of the eukaryotic K(+) channels studied previously, and that the off-rate increases with the applied transmembrane voltage.

Rapid intracellular TEA block of the KcsA potassium channel.

Intracellular tetraethylammonium (TEA) inhibition was studied at the single-channel level in the KcsA potassium channel reconstituted in planar lipid bilayers. TEA acts as a fast blocker (resulting in decreased current amplitude) with an affinity in the 75 mM range even at high bandwidth. Studies over a wide voltage range reveal that TEA block has a complex voltage-dependence that also depends on the ionic conditions.

Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel.

Here we describe the initial functional characterization of a cyclic nucleotide regulated ion channel from the bacterium Mesorhizobium loti and present two structures of its cyclic nucleotide binding domain, with and without cAMP. The domains are organized as dimers with the interface formed by the linker regions that connect the nucleotide binding pocket to the pore domain. Together, structural and functional data suggest the domains form two dimers on the cytoplasmic face of the channel.

Revisiting voltage-dependent relief of block in ion channels: a mechanism independent of punchthrough.

Voltage dependence of block was investigated in a simple model for permeation in a multiion pore. Internal blocker could bind to three states of the open channel that differed in the locations and number of permeant ion bound; blocker dissociation occurs exclusively to the internal solution, and the blocker does not itself enter the electric field. By changing the relative stability of blocker binding to these three states, block displayed voltage dependence with relief of block at high potentials.

Functional influence of the pore helix glutamate in the KcsA K+ channel.

The E71 residue is buried near the selectivity filter in the KcsA K+ channel and forms a carboxyl-carboxylate bridge with D80. We have investigated the importance of E71 by examining neutralization mutants at this position using biochemical and electrophysiological methods. E71 mutations differentially destabilize the detergent-solubilized tetramer; among them, the E71V neutralization mutant has a relatively subtle effect.

Opening the KcsA K+ channel: tryptophan scanning and complementation analysis lead to mutants with altered gating.

The properties of the KcsA channel were investigated using a combination of tryptophan scanning of the two transmembrane helices followed by random mutagenesis at targeted residues. The tryptophan mutants were subjected to two screens: oligomeric stability and ability to complement the K+ uptake deficiency of the TK2420 Escherichia coli strain. Oligomeric stability is affected primarily by mutations at sites that border on and interact with the selectivity filter, while the complementation assays identified residues at the crossing point of the inner helices.