Refinement of the binding site and mode of action of the anticonvulsant Retigabine on KCNQ K+ channels.

The discovery of retigabine has provided access to alternative anticonvulsant compounds with a novel mode of action. Acting as potassium channel opener, retigabine exclusively activates neuronal KCNQ-type K(+) channels, mainly by shifting the voltage-dependence of channel activation to hyperpolarizing potentials. So far, only parts of the retigabine-binding site have been described, including Trp-265 and Gly-340 (according to KCNQ3 numbering) within transmembrane segments S5 and S6, respectively. Using a refined chimeric strategy, we additionally identified a Leu-314 within the pore region of KCNQ3 as crucial for the retigabine effect. Both Trp-265 and Leu-314 are likely to interact with the retigabine molecule, representing the upper and lower margins of the putative binding site. Guided by a structural model of KCNQ3, which was constructed based on the Kv1.2 crystal structure, further residues affecting retigabine-binding could be proposed and were experimentally verified as mediators for the action of the compound. These results strongly suggest that, besides Trp-265 and Leu-314, it is highly likely that another S5 residue, Leu-272, which is conserved in all KCNQ subunits, contributes to the binding site in KCNQ3. More importantly, Leu-338, extending from S6 of the neighboring subunit is also apparently involved in lining the hydrophobic binding pocket for the drug. This pocket, which is formed at the interface of two adjacent subunits, may be present only in the open state of the channel, consistent with the idea that retigabine stabilizes an open-channel conformation.

Immune-related proteins induced in the hemolymph after aseptic and septic injury differ in honey bee worker larvae and adults.

We have employed the proteomic approach in combination with mass spectrometry to study the immune response of honey bee workers at different developmental stages. Analysis of the hemolymph proteins of noninfected, mock-infected and immune-challenged individuals by polyacrylamide gel electrophoresis showed differences in the protein profiles. We present evidence that in vitro reared honey bee larvae respond with a prominent humoral reaction to aseptic and septic injury as documented by the transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin. In contrast, young adult worker bees react with a broader spectrum of immune reactions that include the activation of prophenoloxidase and humoral immune responses. At least seven proteins appeared consistently in the hemolymph of immune-challenged bees, three of which are identical to the AMPs induced also in larvae. The other four, i.e., phenoloxidase (PO), peptidoglycan recognition protein-S2, carboxylesterase (CE), and an Apis-specific protein not assigned to any function (HP30), are induced specifically in adult bees and, with the exception of PO, are not expressed after aseptic injury. Structural features of CE and HP30, such as classical leucine zipper motifs, together with their strong simultaneous induction upon challenge with bacteria suggest an important role of the two novel bee-specific immune proteins in response to microbial infections.