Native gating behavior of ion channels in neurons with null-deviation modeling.

Computational modeling has emerged as an indispensable approach to resolve and predict the intricate interplay among the many ion channels underlying neuronal excitability. However, simulation results using the classic formula-based Hodgkin-Huxley (H-H) model or the superior Markov kinetic model of ion channels often deviate significantly from native cellular signals despite using carefully measured parameters. Here we found that the filters of patch-clamp amplifier not only delayed the signals, but also introduced ringing, and that the residual series resistance in experiments altered the command voltages, which had never been fully eliminated by improving the amplifier itself. To remove all the above errors, a virtual device with the parameters exactly same to that of amplifier was introduced into Markov kinetic modeling so as to establish a null-deviation model. We demonstrate that our novel null-deviation approach fully restores the native gating-kinetics of ion-channels with the data recorded at any condition, and predicts spike waveform and firing patterns clearly distinctive from those without correction.

Dissecting a central flip-flop circuit that integrates contradictory sensory cues in C. elegans feeding regulation.

Feeding behaviour is modulated by both environmental cues and internal physiological states. Appetite is commonly boosted by the pleasant smell (or appearance) of food and destroyed by a bad taste. In reality, animals sense multiple environmental cues at the same time and it is not clear how these sensory inputs are integrated and a decision is made to regulate feeding behaviour accordingly. Here we show that feeding behaviour in Caenorhabditis elegans can be either facilitated by attractive odours or suppressed by repellents. By identifying mutants that are defective for sensory-mediated feeding regulation, we dissected a central flip-flop circuit that integrates two contradictory sensory inputs and generates bistable hormone output to regulate feeding behaviour. As feeding regulation is fundamental to animal survival, we speculate that the basic organizational logic identified here in C. elegans is likely convergent throughout different phyla.

Observing GLUT4 translocation in live L6 cells using quantum dots.

The glucose transporter 4 (GLUT4) plays a key role in maintaining whole body glucose homeostasis. Tracking GLUT4 in space and time can provide new insights for understanding the mechanisms of insulin-regulated GLUT4 translocation. Organic dyes and fluorescent proteins were used in previous studies for investigating the traffic of GLUT4 in skeletal muscle cells and adipocytes. Because of their relative weak fluorescent signal against strong cellular autofluorescence background and their fast photobleaching rate, most studies only focused on particular segments of GLUT4 traffic. In this study, we have developed a new method for observing the translocation of GLUT4 targeted with photostable and bright quantum dots (QDs) in live L6 cells. QDs were targeted to GLUT4myc specifically and internalized with GLUT4myc through receptor-mediated endocytosis. Compared with traditional fluorescence dyes and fluorescent proteins, QDs with high brightness and extremely photostability are suitable for long-term single particle tracking, so individual GLUT4-QD complex can be easily detected and tracked for long periods of time. This newly described method will be a powerful tool for observing the translocation of GLUT4 in live L6 cells.

[Evaluation of heart impact in the 100 m extreme intensity sport using near-infrared non-invasive muscle oxygen detecting device and sports heart rate detection technology].

Using continuous two wavelength near-infrared technology to detect the variation in the consistency of oxygen hemoglobin in the muscle and the sports heart rate wireless real time collection technology, we devised the real time muscle tissue oxygenation and instantaneous heart rate experiment scheme and implemented it for the process of the 100 m run with two parameters given simultaneously. The experiment shows that the concentration of the oxygen hemoglobin in the muscle tissue continues decreasing after the end of the 100 m run, and the time interval between the moment when the concentration of the oxygen hemoglobin attains the minimum value and the moment when the athletes finish the 100 m run is (6.65 +/- 1.10) sec; while the heart rate continues increasing after the end of the 100 m run, and the time interval between the moment when the heart rate attains the maximum value and the moment when the athletes finish the 100 m run is (8.00 +/- 1.57) sec. The results show that the two wavelength near-infrared tissue oxygenation detection technology and the sports heart rate real time collection equipment can accurately measure the sports tissue oxygenation and the heart rate in the extreme intensity sport, and reveal the process of muscle oxygen transportation and consumption and its dynamic character with the heart rate in the extreme intensity sport.

Error analysis of Cm measurement under the whole-cell patch-clamp recording.

We present a method for analysing propagation errors in membrane capacitance (C(m)) measurements under the whole-cell patch-clamp configuration, which mainly focusses on errors in C(m) estimates due to the 'residual' fast capacitance (DeltaC(p)). The method employs a quasi-phasor diagram for visualisation of the analysis. Our results show that both under- and over-compensation of fast capacitance will cause errors in C(m), and errors in the magnitude and in the phase angle of cell admittance make their respective and opposite contributions to propagation errors in C(m). Within optimal frequencies, over-compensation of fast capacitance will cause a smaller propagation error in C(m) and produce more accurate C(m) estimates than under-compensation of fast capacitance will do. Information about how other cell parameters, such as smaller series resistance, baseline C(m) value and the stimulus frequency, change the total error in C(m) due to DeltaC(p) is also provided. Guidelines for accurate C(m) recordings are given to make it easy for users to perform their own error analysis.

Differential regulation of action potentials by inactivating and noninactivating BK channels in rat adrenal chromaffin cells.

Large-conductance Ca(2+)-activated K(+) (BK) channels can regulate cellular excitability in complex ways because they are able to respond independently to two distinct cellular signals, cytosolic Ca(2+) and membrane potential. In rat chromaffin cells (RCC), inactivating BK(i) and noninactivating (BK(s)) channels differentially contribute to RCC action potential (AP) firing behavior. However, the basis for these differential effects has not been fully established. Here, we have simulated RCC action potential behavior, using Markovian models of BK(i) and BK(s) current and other RCC currents. The analysis shows that BK current influences both fast hyperpolarization and afterhyperpolarization of single APs and that, consistent with experimental observations, BK(i) current facilitates repetitive firing of APs, whereas BK(s) current does not. However, the key functional difference between BK(i) and BK(s) current that accounts for the differential firing is not inactivation but the more negatively shifted activation range for BK(i) current at a given [Ca(2+)].

Labeling and imaging of GLUT4 in live L6 cells with quantum dots.

GLUT4 is sequestered in intracellular storage compartments in a basal state and is rapidly translocated to the cell surface in response to insulin stimulation. Regulation of GLUT4 distribution is key for maintaining whole-body glucose homeostasis. To investigate the complicated intracellular movement of GLUT4 vesicles and their interactions with organelles in detail, new probes suitable for long-term tracking of cellular events are required. In this study, we introduce for the first time quantum dots (QDs) as a superior probe into the research of the mechanisms of GLUT4 translocation. QDs are light-emitting semiconductor nanoparticles with unique optical and spectroscopic properties, such as broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. Owing to their remarkable photophysical properties and relatively small size, QDs are emerging as an alternative to conventional dyes for fluorescence-based applications. We have developed a procedure for labeling and imaging GLUT4 in live cells with streptavidin-conjugated quantum dot (QD-SA) and demonstrated that QDs contained in cytoplasm have no obvious negative influence on L6 cells. This study provides a sensitive, nontoxic, long-term imaging platform for observing the dynamics and regulated characteristics of GLUT4 transport.

Zf-and-Hsys-based Cm measurement under the whole-cell patch-clamp recording.

Membrane capacitance (C(m)) measurement, which measures and monitors plasma membrane capacitance and its changes of a cell, is one of the most important quantitative techniques and widely used in the studies of exocytosis and endocytosis. We present here a Z(f)-and-H(sys)-based C(m) measurement method (Z-H method) for our self-developed patch-clamp amplifier (PCA). The method adopts the complex impedance of the feedback resistor of the PCA and a transfer function, H(sys), of the main and stimulus paths to generate the impedance of a patch-clamped cell, which extends Gillis' work for C(m) measurements. The chief advantage of the approach is that it does not depend on idealized assumptions about the impedance supplied to the probe input by measuring the frequency responses of the various components of the PCA. H(sys) is generated on "frequency grids" at different standard settings of the PCA, stored and managed by a simple database, and then retrieved for calculation of cell parameters, which results in a high efficiency. An algorithm for obtaining the trans-admittance of the slow capacitance compensation (C-slow) circuitry is provided to improve the accuracy of impedance measurements when the C-slow circuitry is turned on.

Experimental determination of Cm measurement related hardware parameters of the patch-clamp amplifier.

Accurate Cm measurements rely on accurate determination of specific parameters of a patch-clamp amplifier (PCA). Hardware-related parameters, such as the resistance Rf and the stray capacitance Cf of the feedback resistor, the input capacitance Ci, the injection capacitance Cj, and the extra capacitances introduced by the BNC connector, are of significance in the sense of obtaining absolute estimates of cell parameters. In the present paper, a frequency-domain method, or the f-method for simplicity, is put forward to experimentally determine the actual values of basic circuit elements for our self-developed PCA. The f-method makes use of sine waves and amplitude/phase measurements instead of the square-wave responses to determine the above parameters of a PCA, and thereby calibrates the PAC for capacitance measurements. Experimental results prove that the f-method is excellent in determining hardware-related parameters, with 3-5% error of the impedance of the "10 MOmega setting", and about 2% error of the impedance of the "model cell" of the model circuit for our PCA. The f-method enables us not only to picture components of fast capacitances, but also to guarantee complete fast capacitance compensation; it may be applicable for other PCAs.

Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action.

Insulin-stimulated GLUT4 translocation is central to glucose homeostasis. Functional assays to distinguish individual steps in the GLUT4 translocation process are lacking, thus limiting progress toward elucidation of the underlying molecular mechanism. Here we have developed a robust method, which relies on dynamic tracking of single GLUT4 storage vesicles (GSVs) in real time, for dissecting and systematically analyzing the docking, priming, and fusion steps of GSVs with the cell surface in vivo. Using this method, we have shown that the preparation of GSVs for fusion competence after docking at the surface is a key step regulated by insulin, whereas the docking step is regulated by PI3K and its downstream effector, the Rab GAP AS160. These data show that Akt-dependent phosphorylation of AS160 is not the major regulated step in GLUT4 trafficking, implicating alternative Akt substrates or alternative signaling pathways downstream of GSV docking at the cell surface as the major regulatory node.

[A data interface based on USB bus technology for full auto patch-clamp system].

A USB bus based data interface technology for full auto Patch-Clamp system is discussed in the article. The main controller is CY2131QC (Cypress) and the logic controller is EPM3256A (Altera). Optocouplers are used to get rid of the noise from the interface. It makes the installation of the Patch-Clamp system easier by using the USB bus, and is suitable for the new generation of the Patch-Clamp system with a high speed of 1M bytes/s.

Kir6.2DeltaC26 channel traffics to plasma membrane by constitutive exocytosis.

Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels regulate many cellular functions by coupling the metabolic state of the cell to the changes in membrane potential. Truncation of C-terminal 26 amino acid residues of Kir6.2 protein (Kir6.2DeltaC26) deletes its endoplasmic reticulum retention signal, allowing functional expression of Kir6.2 in the absence of sulfonylurea receptor subunit. pEGFP-Kir6.2DeltaC26 and pKir6.2DeltaC26-IRES2-EGFP expression plasmids were constructed and transfected into HEK293 cells. We identified that Kir6.2DeltaC26 was localized on the plasma membrane and trafficked to the plasmalemma by means of constitutive exocytosis of Kir6.2DeltaC26 transport vesicles, using epi-fluorescence and total internal reflection fluorescence microscopy. Our electrophysiological data showed that Kir6.2DeltaC26 alone expressed KATP currents, whereas EGFP-Kir6.2DeltaC26 fusion protein displayed no KATP channel activity.

[Design of software for fluorescence microscopy system to measure cytosolic free calcium in living cell].

OBJECTIVE: To design software based on the double-wavelength fluorescence microscopy to measure cytosolic free calcium ([Ca2+]i) in living cell. METHOD: Through analyzing the work principles of monochromator, the function relation between output wavelength and voltage was obtained. Based on the analysis results as the arithmetic of the software, the application was developed in the integration development environment of VC ++ 6.0, by using the framework of Microsoft Foundation Class Library (MFC) and multithread technology. RESULT: The authors has realized the software and constructed a fluorescence microscopy system using the software, PC, monochromator, fluorescence microscope, MPT and data acquisition to measure the [Ca2+]i in single living beta cell. CONCLUSION: Using this software, the [Ca2+]i in living cell can be detected. It has been proved to be an effective tool for the research in cellular biophysics.

Designing a Signal Conditioning System with Software Calibration for Resistor-feedback Patch Clamp Amplifier.

In this paper, a programmable signal conditioning system based on software calibration for resistor-feedback patch clamp amplifier (PCA) has been described, this system is mainly composed of frequency correction, programmable gain and filter whose parameters are configured by software automatically to minimize the errors, A lab-designed data acquisition system (DAQ) is used to implement data collections and communications with PC. The laboratory test results show good agreement with design specifications.

Three-Dimensional Tracking of Single Granules in Living PC-12 Cells Employing TIRFM and WFFM.

A comparative study was carried out on evaluating the performance of total internal reflection fluorescence microscopy (TIRFM) and deconvolution wide-field fluorescence microscopy (WFFM) in tracking single secretory granules. Both techniques have been applied to follow the three-dimensional mobility of single secretory granules in living neuroendocrine PC-12 cells. Both techniques return the similar result that most acridine orange-labeled granules were found to travel in random and caged diffusion, and only a small fraction of granules traveled in directed diffusion. Furthermore, the size and 3-D diffusion coefficient of secretory granules, obtained by these two imaging techniques, yield the same value. Together, our results demonstrate the potential of the combination TIRFM and WFFM in tracking long-termed motion of granules throughout live whole cells.

Three-dimensional tracking of single secretory granules in live PC12 cells.

Deconvolution wide-field fluorescence microscopy and single-particle tracking were used to study the three-dimensional mobility of single secretory granules in live PC12 cells. Acridine orange-labeled granules were found to travel primarily in random and caged diffusion, whereas only a small fraction of granules traveled in directed fashion. High K(+) stimulation increased significantly the percentage of granules traveling in directed fashion. By dividing granules into the near-membrane group (within 1 microm from the plasma membrane) and cytosolic group, we have revealed significant differences between these two groups of granules in their mobility. The mobility of these two groups of granules is also differentially affected by disruption of F-actin, suggesting different mechanisms are involved in the motion of the two groups of granules. Our results demonstrate that combined deconvolution and single-particle tracking may find its application in three-dimensional tracking of long-term motion of granules and elucidating the underlying mechanisms.

[Use of three-dimensional fluorescence deconvolution microscopy for study of spatial distribution of secretory vesicles in living cells].

A three-dimensional image of a living cell is helpful for cell secretion study. In this report, the three-dimensional fluorescence deconvolution microscopy for observing living cells was studied, because this technique can obtain a quick three-dimensional imaging with minimal fluorescence quenching and cytotoxicity for living cell observation. The property of three-dimensional point spread function (PSF) of imaging system was analyzed. The relationship between experimental and theoretical PSF was illustrated, and the theoretical PSF was proved that it could reflect the principle of imaging system with NA 1.65 objective in use. Three-dimensional deconvolution algorithm in this report was proved effective by well-defined three-dimensional specimens. Furthermore, the rat pancreatic beta cell secretory vesicles labeled by acridine orange was observed by using this technique. Results showed that the blurring induced by out-of-focus light was removed by the deconvolution algorithm effectively, under current experiment conditions (with NA 1.65 objective) the experimental PSF approximated the theoretical PSF very well, and deconvolved living cell images exhibited the spatial distribution of the secretory vesicles clearly.

Na+ channel inactivation: a comparative study between pancreatic islet beta-cells and adrenal chromaffin cells in rat.

A comparative study was carried out on the inactivation of Na+ channels in two types of endocrine cells in rats, beta-cells and adrenal chromaffin cells (ACCs), using patch-clamp techniques. The beta-cells were very sensitive to hyperpolarization; the Na+ currents increased ninefold when the holding potential was shifted from -70 mV to -120 mV. ACCs were not sensitive to hyperpolarization. The half-inactivation voltages were -90 mV (rat beta-cells) and -62 mV (ACCs). The time constant for recovery from inactivation at -70 mV was 10.5 times slower in beta-cells (60 ms) than in ACCs (5.7 ms). The rate of Na+-channel inactivation at physiological resting potential was more than three times slower in beta-cells than in ACCs. Na+ influx through Na+ channels had no effect on the secretory machinery in rat beta-cells. However, these 'silent Na+ channels' could contribute to the generation of action potentials in some conditions, such as when the cell is hyperpolarized. It is concluded that the fractional availability of Na+ channels in beta-cells at a holding potential of -70 mV is about 15 % of that in ACCs. This value in rat beta-cells is larger than that observed in mouse (0 %), but is smaller than those observed in human or dog (90 %).