Transcending Markov: non-Markovian rate processes of thermosensitive TRP ion channels
Abstract:The Markov state model (MSM) is a popular theoretical tool for describing the hierarchy of time scales involved in the function of many proteins especially ion channel gating. An MSM is a particular case of the general non-Markovian model, where the rate of transition from one state to another does not depend on the history of state occupancy within the system, i.e. it only includes reversible, non-dissipative processes. However, an MSM requires knowledge of the precise conformational state of the protein and is not predictive when those details are not known. In the case of ion channels, this simple description fails in real (non-equilibrium) situations, for example when local temperature changes, or when energy losses occur during channel gating. Here, we show it is possible to use non-Markovian equations (i.e. offer a general description that includes the MSM as a particular case) to develop a relatively simple analytical model that describes the non-equilibrium behaviour of the temperature-sensitive transient receptor potential (TRP) ion channels, TRPV1 and TRPM8. This model accurately predicts asymmetrical opening and closing rates, infinite processes and the creation of new states, as well as the effect of temperature changes throughout the process. This approach therefore overcomes the limitations of the MSM and allows us to go beyond a mere phenomenological description of the dynamics of ion channel gating towards a better understanding of the physics underlying these processes.
Action of the general anaesthetic isoflurane reveals coupling between viscoelasticity and electrophysiological activity in individual neurons
Abstract:General anaesthetics are widely used for their analgesic, immobilising, and hypnotic effects. The mechanisms underlying these effects remain unclear, but likely arise from alterations to cell microstructure, and potentially mechanics. Here we investigate this hypothesis using a custom experimental setup combining calcium imaging and nanoindentation to quantify the firing activity and mechanical properties of dorsal root ganglion-derived neurons exposed to a clinical concentration of 1% isoflurane gas, a halogenated ether commonly used in general anaesthesia. We found that cell viscoelasticity and functional activity are simultaneously and dynamically altered by isoflurane at different stages of exposure. Particularly, cell firing count correlated linearly with the neuronal loss tangent, the ratio of mechanical energy dissipation and storage by the cell. Our results demonstrate that anaesthetics affect cells as a whole, reconciling seemingly contradictory theories of how anaesthetics operate, and highlight the importance of considering cell mechanics in neuronal functions, anaesthesia, and clinical neuroscience in general.
Electrochemical and nanostructural characterization of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) films as coatings for neural electrodes
Abstract:Poly(3,4-ethylenedioxythiophene) (PEDOT), a well-characterized conducting polymer, has been applied for coating metal neural electrodes to improve their stimulating or recording performance. The coated electrodes possess advantages in better neuron attachment, lower impedance, and larger capacitance compared to the bare metal substrate due to the biocompatibility and porous surface of the polymer. However, the PEDOT-coated electrodes have frequently reported issues associated with mechanical instability, such as cracking and delamination. Solving this problem is crucial for stimulating electrodes, whereas a massive film is unnecessary for recording purposes. Moreover, the thickness control for the latter has rarely been investigated. In this work, we systematically studied and characterized poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) with cyclic voltammetry and atomic force microscopy (AFM) to evaluate the electropolymerization of PEDOT:PSS from the basis and analyze the surface morphology for a range of deposition times. The polymerization potential was obtained, and the deposition charge density was optimized for recording neural electrodes. In addition, high-resolution AFM height and phase images reveal the heterogeneity of the polymer surface. The modified electrode was also tested for its electrochemical performance in a small potential window with both a standard electrochemical cell setup and stainless steel microscrews. The results showed that despite a shift of potential (0.42 V) due to the change of setup, the electrode functions well in the capacitive region without triggering redox reactions.
Cell-wall fucosylation in Arabidopsis influences control of leaf water loss and alters stomatal development and mechanical properties
The Arabidopsis sensitive-to-freezing8 (sfr8) mutant exhibits reduced cell wall (CW) fucose levels and compromised freezing tolerance. To examine whether CW fucosylation also affects the response to desiccation, we tested the effect of leaf excision in sfr8 and the allelic mutant mur1-1. Leaf water loss was strikingly higher than in the wild type in these, but not other, fucosylation mutants. We hypothesized that reduced fucosylation in guard cell (GC) walls might limit stomatal closure through altering mechanical properties. Multifrequency atomic force microscopy (AFM) measurements revealed a reduced elastic modulus (Eʹ), representing reduced stiffness, in sfr8 GC walls. Interestingly, however, we discovered a compensatory mechanism whereby a concomitant reduction in the storage modulus (Eʹʹ) maintained a wild-type viscoelastic time response (tau) in sfr8. Stomata in intact leaf discs of sfr8 responded normally to a closure stimulus, abscisic acid, suggesting that the time response may relate more to closure properties than stiffness does. sfr8 stomatal pore complexes were larger than those of the wild type, and GCs lacked a fully developed cuticular ledge, both potential contributors to the greater leaf water loss in sfr8. We present data that indicate that fucosylation-dependent dimerization of the CW pectic domain rhamnogalacturonan-II may be essential for normal cuticular ledge development and leaf water retention.