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Activation Kinetics (activation + kinetics)
Selected AbstractsRegulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2006Rainer Surges Abstract The hyperpolarization-activated current (Ih) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates Ih conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of Ih in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. Ih amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of Ih to cAMP, and the contribution of the slow component to the overall Ih, decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents. [source] Molecular analysis of the A322D mutation in the GABAA receptor ,1 -subunit causing juvenile myoclonic epilepsyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2005Klaus Krampfl Abstract Juvenile myoclonic epilepsy (JME) belongs to the most common forms of hereditary epilepsy, the idiopathic generalized epilepsies. Although the mode of inheritance is usually complex, mutations in single genes have been shown to cause the disease in some families with autosomal dominant inheritance. The first mutation in a multigeneration JME family has been recently found in the ,1 -subunit of the GABAA receptor (GABRA1), predicting the single amino acid substitution A322D. We further characterized the functional consequences of this mutation by coexpressing ,1 -, ,2 - and ,2 -subunits in human embryonic kidney (HEK293) cells. By using an ultrafast application system, mutant receptors have shown reduced macroscopic current amplitudes at saturating GABA concentrations and a highly reduced affinity to GABA compared to the wild-type (WT). Dose,response curves for current amplitudes, activation kinetics, and GABA-dependent desensitization parameters showed a parallel shift towards 30- to 40-fold higher GABA concentrations. Both deactivation and resensitization kinetics were considerably accelerated in mutant channels. In addition, mutant receptors labelled with enhanced green fluorescent protein (EGFP) were not integrated in the cell membrane, in contrast to WT receptors. Therefore, the A322D mutation leads to a severe loss-of-function of the human GABAA receptor by several mechanisms, including reduced surface expression, reduced GABA-sensitivity, and accelerated deactivation. These molecular defects could decrease and shorten the resulting inhibitory postsynaptic currents (IPSCs) in vivo, which can induce a hyperexcitability of the postsynaptic membrane and explain the occurrence of epileptic seizures. [source] Reversible protein kinase C activation in PC12 cells: effect of NGF treatmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2000Jean-Luc Dupont Abstract Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura-2-AM and Fim-1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose-dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl-induced PKC responses was larger indicating an increase in the activated PKC-pool in these cells. This difference between control and NGF-treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+ -dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized. [source] The crystal structure of pyruvate decarboxylase from Kluyveromyces lactisFEBS JOURNAL, Issue 18 2006Implications for the substrate activation mechanism of this enzyme The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 Å resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106,113 and 292,301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105,113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105,113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase. [source] Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissuesFEBS JOURNAL, Issue 6 2001Sven Moosmang Hyperpolarization-activated cation currents (Ih) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN1,4) have been cloned recently. Native Ih currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of Ih currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1,4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1,4 channels expressed in HEK293 cells. All four channel types gave rise to Ih currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native Ih currents is generated, at least in part, by the tissue-specific expression of HCN channel genes. [source] Transport characteristics of N -acetyl- l -aspartate in rat astrocytes: involvement of sodium-coupled high-affinity carboxylate transporter NaC3/NaDC3-mediated transport systemJOURNAL OF NEUROCHEMISTRY, Issue 3 2005Takuya Fujita Abstract We investigated in the present study the transport characteristics of N -acetyl- l -aspartate in primary cultures of astrocytes from rat cerebral cortex and the involvement of NA+ -coupled high-affinity carboxylate transporter NaC3 (formerly known as NaDC3) responsible for N -acetyl- l -aspartate transport. N -acetyl- l -aspartate transport was NA+ -dependent and saturable with a Michaelis,Menten constant (Km) of ,110 µm. NA+ -activation kinetics revealed that the NA+ to- N -acetyl- l -aspartate stoichiometry was 3 : 1 and concentration of Na+ necessary for half-maximal transport (KNAm) was 70 mm. NA+ -dependent N -acetyl- l -aspartate transport was competitively inhibited by succinate with an inhibitory constant (Ki) of 14.7 µm, which was comparable to the Km value of NA+ -dependent succinate transport (29.4 µm). l -Aspartate also inhibited NA+ -dependent [14C]N -acetyl- l -aspartate transport with relatively low affinity (Ki = 2.2 mm), whereas N -acetyl- l -aspartate was not able to inhibit NA+ -dependent aspartate transport in astrocytes. In addition, Li+ was found to have a significant inhibitory effect on the NA+ -dependent N -acetyl- l -aspartate transport in a concentration-dependent manner. Furthermore, RT,PCR and western blot analyses revealed that NaC3 is expressed in primary cultures of astrocytes. Taken collectively, these results indicate that NaC3 expressed in rat cerebrocortical astrocytes is responsible for NA+ -dependent N -acetyl- l -aspartate transport. This transporter is likely to be an essential prerequisite for the metabolic role of N -acetyl- l -aspartate in the process of myelination. [source] In vitro characterization of HCN channel kinetics and frequency dependence in myocytes predicts biological pacemaker functionalityTHE JOURNAL OF PHYSIOLOGY, Issue 7 2009Xin Zhao The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1,5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers. [source] Hyperpolarization-activated cyclic nucleotide-modulated ,HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cellsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Yang Qu The hyperpolarization-activated cation current (If), and the hyperpolarization-activated cyclic nucleotide-modulated ,HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. If and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing If, and the density of If in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The If blocker ZD7288 (0.3 ,m) reduced If and the beating rate of embryoid bodies. Taken together, the activation kinetics of If and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of If to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation. [source] Determinants of activation kinetics in mammalian hyperpolarization-activated cation channelsTHE JOURNAL OF PHYSIOLOGY, Issue 1 2001Takahiro M. Ishii 1The structural basis for the different activation kinetics of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels was investigated with the whole-cell patch clamp technique by using HCN1, HCN4, chimeric channels and mutants in a mammalian expression system (COS,7). 2The activation time constant of HCN4 was about 40-fold longer than that of HCN1 when compared at ,100 mV. 3In chimeras between HCN1 and HCN4, the region of the S1 transmembrane domain and the exoplasmic S1-S2 linker markedly affected the activation kinetics. The cytoplasmic region between S6 and the cyclic nucleotide-binding domain (CNBD) also significantly affected the activation kinetics. 4The S1 domain and S1-S2 linker of HCN1 differ from those of HCN4 at eight amino acid residues, and each single point mutation of them changed the activation kinetics less than 2-fold. However, the effects of those mutations were additive and the substitution of the whole S1 and S1-S2 region of HCN1 by that of HCN4 resulted in a 10, to 20-fold slowing. 5The results indicate that S1 and S1-S2, and S6-CNBD are the crucial components for the activation gating of HCN channels. [source] | ||||||||||