Slow Time Constants Research Articles

Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells.

Association between Hsp90 and the ClC-2 chloride channel upregulates channel function

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl-, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl- concentration [Cl-]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl-]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.

Desensitization of nicotine acetylcholine receptors: Modulation by kinase activation and phosphatase inhibition

The desensitization of α-bungarotoxin-insensitive native neuronal nicotinic receptors was studied in rat cortical cell cultures using the patch clamp technique. Thirty-minute perfusions of nicotine reduced currents evoked by short test pulses of 300 μM acetylcholine over a range of 3 to 300 nM, with an IC 50 of 51 nM. The time course of desensitization onset was fit by a biexponential function consisting of a fast time constant of about 1 min and a slower component of 6–10 min. The desensitization recovery process was also biexponential and was dominated by a slow time constant of 12–20 min, as well as a minor component of about 1 min. The intracellular dialysis of either the protein kinase C activator phorbol-12-myristate-13 acetate or the phosphatase inhibitor cyclosporin A accelerated the desensitization recovery rate by 2-fold. The data imply that endogenous cortical nicotinic receptor channels may enter one of two desensitization states. The first state (D 1) is characterized by rapid entry and recovery, whereas transitions into and out of the second state (D 2) occur at slower rates. The D 2 receptor state may arise by a sequential transition from the D 1 conformation. Protein kinase C activation or phosphatase 2B inhibition may favor the D 1 receptor state over that of D 2 to promote faster overall rates of desensitization recovery.

Ultrafast Dynamics of Liquid Poly(ethylene glycol)s and Crown Ethers Studied by Femtosecond Raman-Induced Kerr Effect Spectroscopy

Ultrafast molecular dynamics of liquid poly(ethylene glycol)s, tetra(ethylene glycol), penta(ethylene glycol), and poly(ethylene glycol) with the molecular weight of 600, and crown ethers, 12-crown-4 and 15-crown-5, have been investigated by means of femtosecond optical heterodyne-detected Raman-induced Kerr effect spectroscopy. Picosecond Kerr transients of poly(ethylene glycol)s and crown ethers are characterized by a biexponential function with the time constants of about 2 and 20 ps. Both the faster and slower time constants do not vary much among the five oligo(ethylene oxide)s. Femtosecond dynamics is discussed based on the Kerr (depolarized Raman) spectra obtained by Fourier transform deconvolution analysis of the high time resolution Kerr transients. The broad low-frequency band (0-200 cm(-1)) in the Kerr spectrum is analyzed by two Brownian oscillators. The spectral shapes of linear poly(ethylene glycol) and cyclic crown ether are very different. Both the low- and high-frequency Brownian oscillators for crown ethers show lower frequency and broader spectral features than those for poly(ethylene glycol)s. The comparison of the low-frequency spectra of poly(ethylene glycol)s and crown ethers shows that the low-frequency spectrum of 15-crown-5 is closer to that of poly(ethylene glycol)s than that of 12-crown-4 is. The difference of the low-frequency spectra between poly(ethylene glycol) and crown ether is discussed with the concepts of molecular conformation and liquid density. The features of the observed intramolecular vibrational bands are also correlated with the molecular conformations.

Ultrafast molecular dynamics of liquid aromatic molecules and the mixtures with CCl4

The ultrafast molecular dynamics of liquid aromatic molecules, benzene, toluene, ethylbenzene, cumene, and 1,3-diphenylpropane, and the mixtures with CCl(4) have been investigated by means of femtosecond optical heterodyne-detected Raman-induced Kerr effect spectroscopy. The picosecond Kerr transients of benzene, toluene, ethylbenzene, and cumene and the mixtures with CCl(4) show a biexponential feature. 1,3-Diphenylpropane and the mixtures with CCl(4) show triexponential picosecond Kerr transients. The slow relaxation time constants of the aromatic molecules and the mixtures with CCl(4) are qualitatively described by the Stoke-Einstein-Debye hydrodynamic model. The ultrafast dynamics have been discussed based on the Kerr spectra in the frequency range of 0-800 cm(-1) obtained by the Fourier transform analysis of the Kerr transients. The line shapes of the low-frequency intermolecular spectra located at 0-180 cm(-1) frequency range have been analyzed by two Brownian oscillators ( approximately 11 cm(-1) and approximately 45 cm(-1) peaks) and an antisymmetric Gaussian function ( approximately 65 cm(-1) peak). The spectrum shape of 1,3-diphenylpropane is quite different from the spectrum shapes of the other aromatic molecules for the low magnitude of the low-frequency mode of 1,3-diphenylpropane and/or an intramolecular vibration. Although the concentration dependences of the low- and intermediate-frequency intermolecular modes (Brownian oscillators) do not show a significant trend, the width of high-frequency intermolecular mode (antisymmetric Gaussian) becomes narrower with the higher CCl(4) concentration for all the aromatics mixtures with CCl(4). The result indicates that the inhomogeneity of the intermolecular vibrational mode in aromatics/CCl(4) mixtures is decreasing with the lower concentration of aromatics. The intramolecular vibrational modes of the aromatic molecules observed in the Kerr spectra are also shown with the calculation results based on the density functional theory.

Antenna-coupled infrared focal plane array

Uncooled bolometric detectors used in infrared-imaging systems have slow time constants (∼15 ms), which makes them impractical for fast-frame-rate applications. Antenna-coupled microbolometers are fast uncooled detectors with good sensitivity and directivity, and they can be polarization and wavelength selective. These detectors have collection areas in the order of 10 μm2, which are too small for infrared imaging systems where a typical pixel area ranges from 20 × 20 μm2 to 50 × 50 μm2. To solve this problem, 2D arrays of antenna-coupled detectors, which can be used as pixels in infrared-imaging systems, are fabricated. In this paper, antenna-coupled pixels are fabricated on a commercial readout integrated circuit (ROIC), resulting in the first antenna-coupled infrared focal plane array (IR-FPA). Measurements made on this antenna-coupled infrared focal plane array show that the integration of antenna-coupled detectors to a commercial ROIC is possible. © 2005 Wiley Periodicals, Inc. Microwave Opt Technol Lett 48: 165–166, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.21294

Transient differential reflectivity of ferromagnetic and paramagnetic phases in the bilayered manganite La 1.24Sr 1.76Mn 2O 7

Photoinduced effects in a single crystal of bilayered manganites, La 2−2 x Sr 1+2 x Mn 2O 7 ( x=0.38), were investigated in a wide range of temperatures by pump-probe measurement at a photon energy of 1.6 eV. In a ferromagnetic metallic state, significant enhancement of positive rise in differential reflectivity with a slow relaxing time of 100 ps was observed just below T C=127 K, indicating that the reflectivity change with the slow relaxation time constant is induced by laser heating. We have also observed an unconventional fast relaxing component that has a time constant of the order of 10 ps. This fast relaxing component, whose absolute value has an asymmetric peak at T C, is presumably due to short-range correlation of Jahn–Teller distortion.

An autocatalytic model of STDP timing from slow calcium-dependent signals

Data of spike timing-dependent plasticity (STDP) show a sharp temporal transition between potentiation and depression despite a relatively slow time course of calcium concentration. We show how autocatalytic amplification of initial concentration differences can enable a high degree of temporal selectivity and produce the sharp STDP weight change curve despite having a relatively slow time constant. This simple model is robust to parameter changes, noise and details of the model. The model correctly predicts the location of the maximum and minimum for STDP at ± 10 ms from coincidence.

Effects of Sodium Bicarbonate on &OV0312;O2 Kinetics during Heavy Exercise

Purpose: Sodium bicarbonate was used to investigate the effect of blood pH on V̇O2 kinetics during heavy exercise. Methods: On separate days, 10 active subjects performed two 6-min cycling bouts (208 ± 12 W) at 25 W above their ventilatory threshold. Each subject ingested 0.3 g·kg−1 of sodium bicarbonate with ∼1 L of water or water alone 1 h before exercise. V̇O2 kinetics were examined by means of a three-component mono-exponential model. Results: Bicarbonate ingestion caused a significant increase in the preexercise blood pH (7.512 ± 0.009 vs 7.425 ± 0.007; P < 0.001). In the bicarbonate trial, the time constant for the rapid component (27.9 ± 3.5 s) was slower than the control trial (20.8 ± 2.4 s; P = 0.017). The higher blood pH after bicarbonate ingestion would have diminished local blood flow and caused a leftward shift of the oxygen-hemoglobin dissociation curve both of which would slow oxygen delivery to working muscle. In addition, bicarbonate ingestion diminished the amplitude of the slow component 29% (463 ± 43 vs 649 ± 53 mL·min−1; P = 0.040). The primary cause of the slow component during heavy exercise is fatigue of working fibers and an accompanying increase of motor unit recruitment. Elevated plasma bicarbonate concentration is reported to stimulate the efflux of H+ from muscle fibers and to increase intramuscular pH. Conclusions: The slower time constant during the rapid component suggested that oxygen delivery is a limiting factor of V̇O2 kinetics during the onset of heavy exercise. Also, these results imply that bicarbonate ingestion diminished fatigue in working fibers during the slow component.

Absorption and emission spectroscopic characterisation of the LOV2-His domain of phot from Chlamydomonas reinhardtii

The absorption and emission behaviour of flavin mononucleotide (FMN) in the wild-type light, oxygen and voltage sensitive domain LOV2 of the photoreceptor phot from the green alga Chlamydomonas reinhardtii is studied. Actually a LOV2-His protein (LOV2 domain bound at N-terminal to 15 His aminoacids via a Gly aminoacid) expressed in an Escherichia coli strain is investigated. For fresh samples stored in the dark an initial fluorescence quantum yield of ϕ F = 0.12 ± 0.01 and an effective fluorescence lifetime of τ F = 2.4 ± 0.1 ns are determined. Blue-light photo-excitation generates an intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm) resulting in an intensity-dependent fluorescence quenching. In the aqueous solutions at pH 8 approximately 3.8% of the FMN molecules are not bound to the protein binding pocket, whereas 96.2% are non-covalently bound. Even at high-intensity light excitation at 428 nm a fraction of about 7% of the non-covalently bound FMN remains non-converted to an FMN-Cys adduct because of photo-induced back-relaxation of the adduct to non-covalently bound FMN. Two holo-LOV2-His conformations with different adduct recovery time constants are revealed by spectrally and temporally resolved fluorescence and absorption measurements: A fraction of about 48% forms FMN-Cys adducts with a fast recovery time constant of τ Ad,f = 19 ± 2 s in the dark, and the rest forms adducts with a slow recovery time constant of τ F,s = 5.5 ± 1 min. Prolonged blue light irradiation of the flavin-C(4a)-cysteinyl adducts reduces their ability to recover back in the dark to non-covalently bound FMN (photo-induced permanent adduct formation). Numerical simulations of the intensity-dependent absorption depletion reveals a quantum yield of intermediate photo-adduct formation of ϕ Ad = 0.9 ± 0.1. Simulation of the adduct absorption dynamics gives a quantum yield of photo-induced adduct back-relaxation of ϕ Ad,b = 0.15 ± 0.01 and a quantum yield of photo-induced permanent adduct formation of ϕ Ad,p = (2.6 ± 0.5) × 10 −4 .

Halothane and propofol modulation of gamma-aminobutyric acidA receptor single-channel currents.

Halothane and propofol enhance the activity of the gamma-aminobutyric acid (GABA) system, which is one of the most important systems in the mechanism of anesthesia. To determine whether halothane and propofol enhance GABAergic responses by the same mechanism, we performed single-channel patch-clamp experiments with rat cortical neurons in primary culture. Each of the open-time and closed-time distributions of GABA(A) receptor single channels was expressed by a sum of fast and slow time constants. Neither halothane nor propofol changed the single-channel conductance. Halothane increased the probability of the channel being open via a prolongation of the slow phase of open time, whereas propofol increased the channel open probability via a shortening of the slow phase of closed time. Thus, although both halothane and propofol augmented the channel open probability, thereby causing an increase in charge transfer during inhibitory transmitter action, they acted by different mechanisms.

A fast photoswitch for minimally perturbed peptides: investigation of the trans-->cis photoisomerization of N-methylthioacetamide.

Thio amino acids can be integrated into the backbone of peptides without significantly perturbing their structure. In this contribution we use ultrafast infrared and visible spectroscopy as well as state-of-the-art ab initio computations to investigate the photoisomerization of the trans form of N-methylthioacetamide (NMTAA) as a model conformational photoswitch. Following the S2 excitation of trans-NMTAA in water, the return of the molecule into the trans ground state and the formation of the cis isomer is observed on a dual time scale, with a fast component of 8-9 ps and a slow time constant of approximately 250 ps. On both time scales the probability of isomerization to the cis form is found to be 30-40%, independently of excitation wavelength. Ab initio CASPT2//CASSCF photochemical reaction path calculations indicate that, in vacuo, the trans-->cis isomerization event takes place on the S1 and/or T1 triplet potential energy surfaces and is controlled by very small energy barriers, in agreement with the experimentally observed picosecond time scale. Furthermore, the calculations identify one S2/S1 and four nearly isoenergetic S1/S0 conical intersection decay channels. In line with the observed isomerization probability, only one of the S1/S0 conical intersections yields the cis conformation upon S1-->S0 decay. A substantially equivalent excited-state relaxation results from four T1/S0 intersystem crossing points.

Chronic NGF treatment of rat nociceptive DRG neurons in culture facilitates desensitization and deactivation of GABAA receptor-mediated currents.

1 The present study tested the hypothesis that nerve growth factor (NGF) could affect presynaptic inhibition mediated by GABAA (GABA-sensitive ionotropic receptors) receptors on the afferents of nociceptive dorsal root ganglia (DRG) neurons, thus reducing the filtering of central nociceptive signals. 2 To investigate this issue, small-diameter, nociceptive DRG neurons were cultured for 48-72 h either in the normal medium or in the presence of NGF (50 ng ml(-1)). After 15 min washout, cells were patch clamped with Cs+ containing electrodes to block GABAB (GABA-sensitive metabotropic receptors) receptor-activated currents. 3 Chronically treated DRG neurons showed no difference in the peak amplitude of GABA-induced currents. However, NGF-treated cells exhibited increased fading of the response to continuous GABA application, with faster desensitization onset, decreased residual current at the end of agonist application and slower recovery from desensitization. Moreover, the deactivation phase after brief agonist pulses was also accelerated. 4 Unlike responses to GABA, chronic NGF treatment had no effect on the desensitization process to the excitatory transmitter ATP, as no difference in peak amplitude, fast and slow time constants of current decay was found. 5 Experimental tests indicated that the observed effects on GABA currents were not a reactive process triggered by washing out NGF after its long application. Acutely applied NGF did not change GABAA receptor-mediated responses. 6 NGF-treated neurons showed decreased sensitivity to the antagonist picrotoxin. The action of pentobarbitone, midazolam, bicuculline or gabazine was, however, unchanged. 7 These observations suggest that the modulation of GABAA receptor function of DRG nociceptors by NGF may contribute to the algogenic action of this neurotrophin.

Development of a total internal reflection ultrafast transient lens method for studying molecular dynamics on an interface.

We have developed the total internal reflection ultrafast transient lens (TIR-UTL) method to detect nonradiative chemical processes at interfaces and surfaces with subpicosecond time resolution. In the TIR-UTL measurements, the evanescent field of a pump beam irradiated under the TIR condition generates a refractive index change. The refractive index change is attributed to changes of the molecular electronic state, of density by molecular orientation/structure change, and of temperature by vibrational relaxation processes. The refractive index change is detected as a change of the power intensity of the probe beam adjusted coaxially with the pump beam. At first, we discuss a theoretical principle of a coaxial configuration in the TIR-UTL measurement. This configuration has an advantage of versatility over the established TIR configuration. Then, we evaluate time resolution of TIR-UTL and obtain a value of less than 400 fs. We measure the ultrafast molecular dynamics of the cationic chromophore Auramine O (AuO) at a silica/water interface. Two slow time constants originating from AuO adsorbed on the silica surface are detected by TIR-UTL. These are attributed to AuO, whose twisting motion is strongly hindered by adsorption on a silica surface.

Does the Onset of Water Droplet Formation Alter the Microenvironment of the Hydrophobic Probes Solubilized in Nonionic Reverse Micelles?

The interiors of the reverse micelles formed with the surfactant Triton X-100 (TX-100) in benzene−n-hexane mixed solvent with an increasing water content, W = [H2O]/[TX-100], have been explored by monitoring the fluorescence anisotropies of two structurally similar but chemically distinct hydrophobic probes. The objective of this work is to find out how the formation of the water pool influences the location and mobility of these probe molecules. It has been observed that the anisotropies of both the probes 2,5-dimethyl-1,4-dioxo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DMDPP) and 1,4-dioxo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DPP) decay as a sum of two exponentials with slow and fast time constants. This experimental finding has been rationalized on the basis of a two-step model according to which the probe molecule undergoes two different kinds of motion inside the micelle. The average reorientation times of DPP are a factor of 4 longer than that of DMDPP. This observation in conjunction with the fact that the ...

G2 chromatid damage and repair kinetics in normal human fibroblast cells exposed to low- or high-LET radiation

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high-LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle.

Effects of salicylate on voltage-gated sodium channels in rat inferior colliculus neurons

To investigate the effects of the tinnitus inducer, sodium salicylate, on voltage-gated sodium channels, we studied freshly dissociated inferior colliculus neurons of rats by the whole-cell voltage clamp method. Salicylate blocked sodium channels in concentration-dependent manner (0.1–10 mM), and the IC 50 value of salicylate was estimated to be 1.43 mM after application. The sodium conductance–voltage curve did not shift along the voltage axis with salicylate application. In contrast, the steady-state sodium channel inactivation curve was shifted by about 9 mV in the hyperpolarizing direction. In addition, salicylate delayed the sodium channel recovery from inactivation by increasing the slow time constant. It was concluded that salicylate bound to the resting and inactivated sodium channels to cause blocking, with a higher affinity for the latter state. Our results suggest that salicylate causes a concentration-dependent blockade of voltage-gated sodium channels and shifts the inactivation curve to more hyperpolarized potentials, which could be related to the mechanism of salicylate-induced tinnitus.

Are the Experimentally Determined Microviscosities of the Micelles Probe Dependent?

With an intent to address the question posed in the title, rotational diffusion of two dissimilar probes, 2,5-dimethyl-1,4-dioxo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DMDPP) and coumarin 6 (C6), has been investigated in small aqueous micelles formed with the surfactants sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), 2-phenyldodecanesulfonate (2-PDS), 3-phenyldodecanesulfonate (3-PDS), 4-phenyldodecanesulfonate (4-PDS), and 6-phenyldodecanesulfonate (6-PDS) using the time-resolved fluorescence depolarization method. The decay of anisotropy for both the probes in all the micelles could be adequately described by a sum of two exponentials with slow and fast time constants. These results have been rationalized on the basis of a two-step model consisting of fast-restricted rotation of the probe and slow lateral diffusion of the probe in the micelle that are coupled to the overall rotation of the micelle. From the order parameter values it has been established that the probes are located in t...

Dynamics of brain activity in motor and frontal cortical areas during music listening: a magnetoencephalographic study

There are formidable problems in studying how ‘real’ music engages the brain over wide ranges of temporal scales extending from milliseconds to a lifetime. In this work, we recorded the magnetoencephalographic signal while subjects listened to music as it unfolded over long periods of time (seconds), and we developed and applied methods to correlate the time course of the regional brain activations with the dynamic aspects of the musical sound. We showed that frontal areas generally respond with slow time constants to the music, reflecting their more integrative mode; motor-related areas showed transient-mode responses to fine temporal scale structures of the sound. The study combined novel analysis techniques designed to capture and quantify fine temporal sequencing from the authentic musical piece (characterized by a clearly defined rhythm and melodic structure) with the extraction of relevant features from the dynamics of the regional brain activations. The results demonstrated that activity in motor-related structures, specifically in lateral premotor areas, supplementary motor areas, and somatomotor areas, correlated with measures of rhythmicity derived from the music. These correlations showed distinct laterality depending on how the musical performance deviated from the strict tempo of the music score, that is, depending on the musical expression.

Ito Channels Are Octomeric Complexes with Four Subunits of Each Kv4.2 and K+ Channel-interacting Protein 2

Mammalian voltage-gated K+ channels are assemblies of pore-forming alpha-subunits and modulating beta-subunits. To operate correctly, Kv4 alpha-subunits in the heart and central nervous system require recently identified beta-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2.KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing beta-subunits.