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 approximately 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 approximately 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.
Ohndorf
Uta-Maria
UM
Howard Hughes Medical Institute, The Laboratory of Molecular Neurobiology and Biophysics, Box 47, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
MacKinnon
Roderick
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001665:Binding Sites; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000737:chemistry; D008051:Lipid Bilayers; D009712:Nucleotides, Cyclic / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology; D011487:Protein Conformation; D015202:Protein Engineering; D048170:Streptomyces lividans / Q000737:chemistry*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D008051:Lipid Bilayers; D009712:Nucleotides, Cyclic; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D000242:Cyclic AMP