Construction of a Cyclic Nucleotide-gated KcsA K+ Channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by ∼50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of ∼90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.