Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K channels, Kcv and Kcv. These small viral encoded K channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in Kcv and 40% in Kcv. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in Kcv. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.
Ion channel proteins can be in vitro translated into nanoscale lipid bilayers known as nanodiscs. Winterstein et al. show that they can subsequently insert into planar bilayers, providing a rapid and contamination-free method for functional characterization.
Background Neuropathic pain is experienced worldwide by patients suffering from nerve injuries, infectious or metabolic diseases or chemotherapy. However, the treatment options are still limited because of low efficacy and sometimes severe side effects. Recently, the deficiency of FKBP51 was shown to relieve chronic pain, revealing FKBP51 as a potential therapeutic target. However, a specific and potent FKBP51 inhibitor was not available until recently which hampered targeting of FKBP51. Methods In this study, we used the well-established and robust spared nerve injury model to analyze the effect of SAFit2 on nerve injury-induced neuropathic pain and to elucidate its pharmacodynamics profile. Therefore, the mice were treated with 10 mg/kg SAFit2 after surgery, the mice behavior was assessed over 21 days and biochemical analysis were performed after 14 and 21 days. Furthermore, the impact of SAFit2 on sensory neurons and macrophages was investigated in vitro. Results Here, we show that the FKBP51 inhibitor SAFit2 ameliorates nerve injury-induced neuropathic pain in vivo by reducing neuroinflammation. SAFit2 reduces the infiltration of immune cells into neuronal tissue and counteracts the increased NF-κB pathway activation which leads to reduced cytokine and chemokine levels in the DRGs and spinal cord. In addition, SAFit2 desensitizes the pain-relevant TRPV1 channel and subsequently reduces the release of pro-inflammatory neuropeptides from sensory neurons. Conclusions SAFit2 ameliorates neuroinflammation and counteracts enhanced neuronal activity after nerve injury leading to an amelioration of nerve injury-induced neuropathic pain. Based on these findings, SAFit2 constitutes as a novel and promising drug candidate for the treatment of nerve injury-induced neuropathic pain.
Lipid bilayers provide many benefits for ion channel recordings, such as control of membrane composition and transport molecules. However, they suffer from high membrane capacitance limiting the bandwidth and impeding analysis of fast gating. This can be overcome by fitting the deviations of the open channel noise from the baseline noise by extended beta distributions. We demonstrate this analysis step-by-step by applying it to the example of viral K+ channels (Kcv), from the choice of the gating model through the fitting process, validation of the results, and what kinds of results can be obtained. These voltage sensor-less channels show profoundly voltage-dependent gating with dwell times in the closed state of about 50 μs. Mutations assign it to the selectivity filter.
Plants acquire potassium (K + ) ions for cell growth and movement via regulated diffusion through K + channels. Here, we present crystallographic and functional data showing that the K + inward rectifier KAT1 (K + Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H + -ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K + uptake in plant cells.
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