Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings.
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ABSTRACT:
SUBMITTER: Williams SR
PROVIDER: S-EPMC3072097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca(2+) on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K(+) (E K), indicating K(+) selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at -80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na(+) and K(+) selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na(+) influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca(2+) revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca(2+) activated SV channels, vacuolar Ca(2+) inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca(2+) was also determined. These channels were active even without Ca(2+) (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca(2+) on the cytoplasmic side. Apart from monovalent cations (K(+) and Na(+)), SV channels were permeable to divalent cations (Ca(2+) and Mg(2+)). Both monovalent and divalent cations passed through the channels in the same direction-from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.
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Project description:Mathematical models of ion channels, which constitute indispensable components of action potential models, are commonly constructed by fitting to whole-cell patch-clamp data. In a previous study, we fitted cell-specific models to hERG1a (Kv11.1) recordings simultaneously measured using an automated high-throughput system, and studied cell-cell variability by inspecting the resulting model parameters. However, the origin of the observed variability was not identified. Here, we study the source of variability by constructing a model that describes not just ion current dynamics, but the entire voltage-clamp experiment. The experimental artefact components of the model include: series resistance, membrane and pipette capacitance, voltage offsets, imperfect compensations made by the amplifier for these phenomena, and leak current. In this model, variability in the observations can be explained by either cell properties, measurement artefacts, or both. Remarkably, by assuming that variability arises exclusively from measurement artefacts, it is possible to explain a larger amount of the observed variability than when assuming cell-specific ion current kinetics. This assumption also leads to a smaller number of model parameters. This result suggests that most of the observed variability in patch-clamp data measured under the same conditions is caused by experimental artefacts, and hence can be compensated for in post-processing by using our model for the patch-clamp experiment. This study has implications for the question of the extent to which cell-cell variability in ion channel kinetics exists, and opens up routes for better correction of artefacts in patch-clamp data. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.
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Project description:The activity of trans-membrane proteins such as ion channels is the essence of neuronal transmission. The currently most accurate method for determining ion channel kinetic mechanisms is single-channel recording and analysis. Yet, the limitations and complexities in interpreting single-channel recordings discourage many physiologists from using them. Here we show that a genetic search algorithm in combination with a gradient descent algorithm can be used to fit whole-cell voltage-clamp data to kinetic models with a high degree of accuracy. Previously, ion channel stimulation traces were analyzed one at a time, the results of these analyses being combined to produce a picture of channel kinetics. Here the entire set of traces from all stimulation protocols are analysed simultaneously. The algorithm was initially tested on simulated current traces produced by several Hodgkin-Huxley-like and Markov chain models of voltage-gated potassium and sodium channels. Currents were also produced by simulating levels of noise expected from actual patch recordings. Finally, the algorithm was used for finding the kinetic parameters of several voltage-gated sodium and potassium channels models by matching its results to data recorded from layer 5 pyramidal neurons of the rat cortex in the nucleated outside-out patch configuration. The minimization scheme gives electrophysiologists a tool for reproducing and simulating voltage-gated ion channel kinetics at the cellular level.
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