5 research outputs found
Vancomycin does not affect the enzymatic activities of purified VanSA.
VanS is a membrane-bound sensor histidine kinase responsible for sensing vancomycin and activating transcription of vancomycin-resistance genes. In the presence of vancomycin, VanS phosphorylates the transcription factor VanR, converting it to its transcriptionally active form. In the absence of vancomycin, VanS dephosphorylates VanR, thereby maintaining it in a transcriptionally inactive state. To date, the mechanistic details of how vancomycin modulates VanS activity have remained elusive. We have therefore studied these details in an in vitro system, using the full-length VanS and VanR proteins responsible for type-A vancomycin resistance in enterococci. Both detergent- and amphipol-solubilized VanSA display all the enzymatic activities expected for a sensor histidine kinase, with amphipol reconstitution providing a marked boost in overall activity relative to detergent solubilization. A putative constitutively activated VanSA mutant (T168K) was constructed and purified, and was found to exhibit the expected reduction in phosphatase activity, providing confidence that detergent-solubilized VanSA behaves in a physiologically relevant manner. In both detergent and amphipol solutions, VanSA's enzymatic activities were found to be insensitive to vancomycin, even at levels many times higher than the antibiotic's minimum inhibitory concentration. This result argues against direct activation of VanSA via formation of a binary antibiotic-kinase complex, suggesting instead that either additional factors are required to form a functional signaling complex, or that activation does not require direct interaction with the antibiotic
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Voltage-Dependent Profile Structures of a Kv-Channel via Time-Resolved Neutron Interferometry
Available experimental techniques cannot determine high-resolution three-dimensional structures of membrane proteins under a transmembrane voltage. Hence, the mechanism by which voltage-gated cation channels couple conformational changes within the four voltage sensor domains, in response to either depolarizing or polarizing transmembrane voltages, to opening or closing of the pore domain's ion channel remains unresolved. Single-membrane specimens, composed of a phospholipid bilayer containing a vectorially oriented voltage-gated K+ channel protein at high in-plane density tethered to the surface of an inorganic multilayer substrate, were developed to allow the application of transmembrane voltages in an electrochemical cell. Time-resolved neutron reflectivity experiments, enhanced by interferometry enabled by the multilayer substrate, were employed to provide directly the low-resolution profile structures of the membrane containing the vectorially oriented voltage-gated K+ channel for the activated, open and deactivated, closed states of the channel under depolarizing and hyperpolarizing transmembrane voltages applied cyclically. The profile structures of these single membranes were dominated by the voltage-gated K+ channel protein because of the high in-plane density. Importantly, the use of neutrons allowed the determination of the voltage-dependent changes in both the profile structure of the membrane and the distribution of water within the profile structure. These two key experimental results were then compared to those predicted by three computational modeling approaches for the activated, open and deactivated, closed states of three different voltage-gated K+ channels in hydrated phospholipid bilayer membrane environments. Of the three modeling approaches investigated, only one state-of-the-art molecular dynamics simulation that directly predicted the response of a voltage-gated K+ channel within a phospholipid bilayer membrane to applied transmembrane voltages by utilizing very long trajectories was found to be in agreement with the two key experimental results provided by the time-resolved neutron interferometry experiments
Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation
Sevoflurane
is a commonly used inhaled general anesthetic. Despite this, its mechanism
of action remains largely elusive. Compared to other anesthetics,
sevoflurane exhibits distinct functional activity. In particular,
sevoflurane is a positive modulator of voltage-gated <i>Shaker</i>-related potassium channels (K<sub>v</sub>1.x), which are key regulators
of action potentials. Here, we report the synthesis and validation
of azisevoflurane, a photoaffinity ligand for the direct identification
of sevoflurane binding sites in the K<sub>v</sub>1.2 channel. Azisevoflurane
retains major sevoflurane protein binding interactions and pharmacological
properties within <i>in vivo</i> models. Photoactivation
of azisevoflurane induces adduction to amino acid residues that accurately
reported sevoflurane protein binding sites in model proteins. Pharmacologically
relevant concentrations of azisevoflurane analogously potentiated
wild-type K<sub>v</sub>1.2 and the established mutant K<sub>v</sub>1.2 G329T. In wild-type K<sub>v</sub>1.2 channels, azisevoflurane
photolabeled Leu317 within the internal S4–S5 linker, a vital
helix that couples the voltage sensor to the pore region. A residue
lining the same binding cavity was photolabeled by azisevoflurane
and protected by sevoflurane in the K<sub>v</sub>1.2 G329T. Mutagenesis
of Leu317 in WT K<sub>v</sub>1.2 abolished sevoflurane voltage-dependent
positive modulation. Azisevoflurane additionally photolabeled a second
distinct site at Thr384 near the external selectivity filter in the
K<sub>v</sub>1.2 G329T mutant. The identified sevoflurane binding
sites are located in critical regions involved in gating of K<sub>v</sub> channels and related ion channels. Azisevoflurane has thus
emerged as a new tool to discover inhaled anesthetic targets and binding
sites and investigate contributions of these targets to general anesthesia