Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+channels as a novel pharmacological target and provides a rational framework for drug design.

Proteins have evolved to balance efficient binding of desired partners with rejection of unwanted interactions. To investigate the evolution of protein-protein interactions, we selected a random library of pre-stabilized TEM1 β-lactamase against wild-type TEM1 using yeast surface display. Three mutations were sufficient to achieve micromolar affinity binding between the two. The X-ray structure emphasized that the main contribution of the selected mutations was to modify the protein fold, specifically removing the N'-terminal helix, which consequently allowed protein coupling via a β-sheet-mediated interaction resembling amyloid interaction mode. The only selected mutation located at the interaction interface (E58V) is reminiscent of the single mutation commonly causing sickle-cell anemia. Interestingly, the evolved mutations cannot be inserted into the wild-type protein due to reduced thermal stability of the resulting mutant protein. These results reveal a simple mechanism by which undesirable binding is purged by loss of thermal stability.

Voltage dependent potassium channels (K v s) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K + -selective pore. Animal toxins targeting K v s are classified to "pore-blockers" that physically plug the ion conduction pathway and "gating modifiers" that disrupt voltage sensor movements. A third group of toxins blocks K + conduction by an unknown mechanism via binding to the channel turrets. Here we show that Cs1, a peptide toxin isolated from cone snail venom, binds at the turrets of K v 1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K + channels as a novel pharmacological target and provides a rational framework for drug design.

Hyperpolarization-activated, Cyclic Nucleotide-gated (HCN) channels are major determinants of the firing rate of pacemaker centers in the heart and brain. Direct binding of cAMP to the cyclic nucleotide binding domain (CNBD) activates HCN channels by increasing the maximum open probability and shifting the voltage-dependence of activation to less hyperpolarized potentials. HCN isoforms differ greatly in kinetics, voltage gating, and response to cyclic nucleotides. HCN2 responds strongly to cAMP despite having similar ligand affinity to HCN1, an isoform that responds very poorly to cAMP. To identify the key molecular determinants responsible for cAMP activation, we progressively mutated sites in the C-linker and CNBD of the mHCN2 to HCN1. Our studies identified two clusters of mutations that determine the differences in voltage dependent activation between these two isoforms. One mapped to the C-linker region (M485F, G497D, and S514T), far from the cAMP-binding site. Another was found in proximity to the binding site (V562A/S563G, L565I, and S575T). Concurrent substitutions of five sites (M485F, G497D, S514T, and V562A/ S563G) is sufficient to confer HCN1 phenotype on the mutated HCN2 channels. To understand the role of these residues on various allosteric parameters that determine the gating of these channels, we built an allosteric model consisting of four separate modules: pore gates, four voltage-sensors, ligand binding domain, and C-linker. Our models suggest that these substitutions must alter at least the oligomerization state of the linker and two coupling parameters: one between the linker and the pore and the other between the linker and the voltage sensors. These findings will be discussed in light of the highresolution structures of HCN channels.

No abstract