Background: TRPA1 channel is modulated by Ca 2ϩ , but the molecular mechanisms are unclear. Results: Mutations in the distal C-terminal acidic domain altered Ca 2ϩ dependence of TRPA1. Conclusion: The C-terminal acidic cluster is involved in the Ca 2ϩ -induced potentiation and inactivation of TRPA1. Significance: Identification of the Ca 2ϩ -dependent domain is important for understanding the role of TRPA1 in chemical nociception.
Transient receptor potential ankyrin 1 (TRPA1) is a temperature-sensitive ion channel activated by various pungent and irritant compounds that can produce pain in humans. Its activation involves an allosteric mechanism whereby electrophilic agonists evoke interactions within cytosolic domains and open the channel pore through an integrated nexus formed by intracellular membrane proximal regions that are densely packed beneath the lower segment of the S1-S4 sensor domain. Studies indicate that this part of the channel may contain residues that form a water-accessible cavity that undergoes changes in solvation during channel gating. We identified conserved polar residues facing the putative lower crevice of the sensor domain that were crucial determinants of the electrophilic, voltage, and calcium sensitivity of the TRPA1 channel. This part of the sensor may also comprise a domain capable of binding to membrane phosphoinositides through which gating of the channel is regulated in a state-dependent manner.
The high sensitivity to pH of a short segment (an octamer) of serum response factor (SRF), an important member of the MADS box family of transcription factors, was investigated by Raman scattering, infrared and circular dichroism spectroscopies. Molecular dynamics (MD) and density functional theory (DFT) calculations enabled interpretation of spectral changes in close detail. Although there was a negligible difference between spectra in acidic and neutral environments, the spectrum in basic pH was substantially different. The major changes were attributed to the deprotonation of tyrosine. The secondary structure of the SRF octamer fragment was estimated experimentally as well as predicted theoretically by MD. All techniques proved that it exists in a dynamical equilibrium among several conformations mostly close to β turn, unordered conformations, and extended structure, in contrast to the stable secondary structure it possesses as a part of SRF. Generally, this approach represents a useful tool for the study of various short oligopeptides.
Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel involved in pain, inflammation and itching. This channel gates in response to many irritant and proalgesic agents, and can be modulated by calcium and depolarizing voltage. While the closed-state structure of TRPA1 has been recently resolved, also having its open state is essential for understanding how this channel works. Here we use molecular dynamics simulations combined with electrophysiological measurements and systematic mutagenesis to predict and explore the conformational changes coupled to the expansion of the presumptive channel's lower gate. We show that, upon opening, the upper part of the sensor module approaches the pore domain of an adjacent subunit and the conformational dynamics of the first extracellular flexible loop may govern the voltage-dependence of multimodal gating, thereby serving to stabilize the open state of the channel. These results are generally important in understanding the structure and function of TRPA1 and offer new insights into the gating mechanism of TRPA1 and related channels.
We have investigated structural changes of peptides related to antimicrobial peptide Halictine-1 (HAL-1) induced by interaction with various membrane-mimicking models with the aim to identify a mechanism of the peptide mode of action and to find a correlation between changes of primary/secondary structure and biological activity. Modifications in the HAL-1 amino acid sequence at particular positions, causing an increase of amphipathicity (Arg/Lys exchange), restricted mobility (insertion of Pro) and consequent changes in antimicrobial and hemolytic activity, led to different behavior towards model membranes. Secondary structure changes induced by peptide-membrane interaction were studied by circular dichroism, infrared spectroscopy, and fluorescence spectroscopy. The experimental results were complemented by molecular dynamics calculations. An α-helical structure has been found to be necessary but not completely sufficient for the HAL-1 peptides antimicrobial action. The role of alternative conformations (such as β-sheet, PPII or 310-helix) also seems to be important. A mechanism of the peptide mode of action probably involves formation of peptide assemblies (possibly membrane pores), which disrupt bacterial membrane and, consequently, allow membrane penetration.
strips placed below. The membrane had 0.4 mm pores that allowed the cytokines released from the CD4þT cells to reach the SM but prevented direct contact between cells. We found a significant increase in Vmax when the SM were in contact with the T cells (0.3250.02 l/s) compared to the co-culture without contact (0.2550.01 l/s) or compared to the SM alone (0.2450.01 l/s; p=0.01). Thus, contact between SM and CD4þT cells is necessary for modulation of SM contractile properties and this may be mediated, at least in part, by increases in (þ)insert myosin heavy chain and MLCK expression. Supported by: CIHR, NIH-RO1HL103405.
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