Non-cooperative ligand binding of a cyclic-nucleotide dependent K+-channel

Cyclic nucleotide-gated (CNG) channels play key roles in signal transduction of sensory neurons. Hyperpolarization-activated cyclic (HCN) channels are important transducers involving signaling pathways in cardiac and neuronal excitability. A common underlying feature of the two channel types is their ability to bind cyclic nucleotides. Both channel types harbour a C-terminal cyclic nucleotide-binding domain. CNG channels select cGMP over cAMP while the HCN channels show greater sensitivity for cAMP. The molecular basis that sets apart ligand affinity and selectivity has not been directly measured but rather inferred from electrophysiological studies. Because binding and gating events are intimately coupled, it is difficult to dissect one from the other.
 
In the following talk, I will present ligand binding studies of a cyclic nucleotide-activated K+ channel from Mesorhizobium loti and its isolated cyclic nucleotide-binding domain. The channel and the isolated binding domain bind cAMP with similar affinity in a non-cooperative fashion. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic CNG channels.
 
Additionally, crystallography experiments were performed on the bacterial channel. Two-dimensional crystals were obtained in which the channel proteins were ordered in a square lattice. The channel proteins were assembled as tetramers and were arranged in a head-to tail fashion. The crystal diffracts to 15 Ang. This served as an excellent starting condition to study the protein structure at atomic level details using solid-state NMR spectroscopy.