Background: HCN2 and HCN4 respond to cAMP, whereas HCN1 does not. Results: The C-linker plus CNBD of HCN2 and HCN4 show cAMP-induced tetramerization, whereas that of HCN1 contains prebound cAMP and is tetrameric. Conclusion: HCN1 does not respond to the addition of cAMP because its CNBD contains cAMP already. Significance: Tetramerization of the C terminus controls ligand gating in HCN channels.

No abstract

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a central role in the regulation of cardiac and neuronal firing rate, and these channels can be dually activated by membrane hyperpolarization and by binding of cyclic nucleotides. cAMP has been shown to directly bind HCN channels and modulate their activity. Despite this, while there are selective inhibitors that block the activation potential of the HCN channels, regulation by cAMP analogs has not been well investigated. A comprehensive screen of 47 cyclic nucleotides with modifications in the nucleobase, ribose moiety, and cyclic phosphate was tested on the three isoforms HCN1, HCN2, and HCN4. 7-CH-cAMP was identified to be a high affinity binder for HCN channels and crosschecked for its ability to act on other cAMP receptor proteins. While 7-CH-cAMP is a general activator for cAMP- and cGMP-dependent protein kinases as well as for the guanine nucleotide exchange factors Epac1 and Epac2, it displays the highest affinity to HCN channels. The molecular basis of the high affinity was investigated by determining the crystal structure of 7-CH-cAMP in complex with the cyclic nucleotide binding domain of HCN4. Electrophysiological studies demonstrate a strong activation potential of 7-CH-cAMP for the HCN4 channel in vivo. So, this makes 7-CH-cAMP a promising activator of the HCN channels in vitro whose functionality can be translated in living cells.

Two open randomized cross-over studies were undertaken comparing ceftazidime to tobramycin and ceftazidime to tobramycin plus carbenicillin in 13 and 15 cystic fibrosis (CF) patients, respectively, with chronic bronchopulmonary Pseudomonas aeruginosa infection. The difference in lung function improvement was statistically better in terms of FEV1 and FVC for the ceftazidime group in the study versus tobramycin plus carbenicillin. Patients receiving ceftazidime showed a tendency for a greater long-term benefit in lung function as measured at 1 and 2 months after treatment than patients receiving the other antibiotics. Development of resistance against both ceftazidime and carbenicillin was seen regularly and was not prevented by combination therapy with tobramycin. No resistance developed when tobramycin was used as monotherapy. Serum concentration curves for ceftazidime fitted a two compartment first order open model in CF patients and showed a distribution volume of 40% of the body weight and a final serum half-life of 1.8 h. One case of Type III hypersensitivity reaction was seen during ceftazidime treatment. Ceftazidime seems to be an effective and safe antibiotic in the treatment of Ps. aeruginosa bronchopulmonary infection in CF patients, although these bacteria could not be eradicated.