Phase Of Inactivation Research Articles

Corrigendum to “Analysis of the variation in use-dependent inactivation of high-threshold tetrodotoxin-resistant sodium currents recorded from rat sensory neurons”

On page 931, Fig. 6, in the Tripathi et al. (2006) study, there was a glitch in the pClamp 8 protocol used to acquire data on inactivation from the closed state of high-threshold tetrodotoxin-resistant Na currents recorded from acutely isolated rat DRG cells. The glitch made it appear as if there was a fast phase of inactivation, which was complete before the first time point of 5 ms included in the protocol. Subsequent experiments with a revised data acquisition protocol demonstrated that no inactivation from the closed state occurred over the first 5 ms at 50 mV, and that overall inactivation was well fitted with a single exponential function. This new information was used to correct the data presented in the results and in Fig. 6 on inactivation from the closed state in the Tripathi et al. (2006) study. The correction method was to subtract out the artifactual inactivation corresponding to the 5 ms time point from that observed at the other time points. The resulting corrected Fig. 6 is presented below. For the corrected data, the time constant for inactivation from the closed state averaged 2667 827 ms, and there was a significant correlation between the amount of inactivation from the closed state observed after 10 s, and use-dependent inactivation (see part D below). Other data acquired in the Tripathi et al. (2006) study indicated that the high threshold tetrodotoxin-resistant Na channels expressed by individual DRG cells were homogeneous regarding inactivation properties regardless of the amount of use-dependent inactivation exhibited (see Fig. 2, Tripathi et al., 2006). Thus, we believe that the method of correction employed is reasonably accurate. The correct Fig. 6 appears here. (Available online 12 May 2011)

Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

The Nav1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Nav1.6 exist predominantly as Nav1.6+β1+β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Nav1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Nav1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.

Cooperativeness of Orai Cytosolic Domains Tunes Subtype-specific Gating

Activation of immune cells is triggered by the Ca2+ release-activated Ca2+ current, which is mediated via channels of the Orai protein family. A key gating process of the three Orai channel isoforms to prevent Ca2+ overload is fast inactivation, most pronounced in Orai3. A subsequent reactivation is a unique gating characteristic of Orai1 channels, whereas Orai2 and Orai3 currents display a second, slow inactivation phase. Employing a chimeric approach by sequential swapping of respective intra- and extracellular regions between Orai1 and Orai3, we show here that Orai1 specific proline/arginine-rich domains in the N terminus mediate reactivation, whereas the second, intracellular loop modulates fast and slow gating processes. Swapping C-terminal strands lacks a significant impact. However, simultaneous transfer of Orai3 N terminus and its second loop or C terminus in an Orai1 chimera substantially increases fast inactivation centered between wild-type channels. Concomitant swap of all three cytosolic strands from Orai3 onto Orai1 fully conveys Orai3-like gating characteristics, in a strongly cooperative manner. In conclusion, Orai subtype-specific gating requires a cooperative interplay of all three cytosolic domains.

Cooperativeness of Orai Cytosolic Domains Tunes Subtype-Specific Gating

Activation of immune cells is triggered by the Ca2+ release-activated Ca2+ (CRAC) current, which is mediated via channels of the Orai protein family. A key gating process of the three Orai channel isoforms to prevent Ca2+ overload is fast inactivation, most pronounced in Orai3. A subsequent reactivation is a unique gating characteristic of Orai1 channels, while Orai2 and Orai3 currents display a second, slow inactivation phase. Employing a chimeric approach by sequential swapping of respective intra- and extra-cellular regions between Orai1 and Orai3, we show here that Orai1 specific proline-arginine-rich domains in the N-terminus mediate reactivation, while the second, intracellular loop modulates fast and slow gating processes. Swapping C-terminal strands lacks a significant impact. However, simultaneous transfer of Orai3 N-terminus and its second loop or C-terminus in an Orai1 chimera substantially increases fast inactivation centered between wild-type channels. Concomitant swap of all three cytosolic strands from Orai3 onto Orai1 fully conveys Orai3-like gating characteristics, in a strongly cooperative manner. In conclusion Orai subtype-specific gating requires a cooperative interplay of all three cytosolic domains.

Rod photoreceptor temporal properties in retinitis pigmentosa

One of the characteristic signs of retinitis pigmentosa (RP) is the progressive loss of night vision. We have previously shown that the gain of rod photoreceptor activation is moderately reduced in some patients with RP, but this decrease in activation kinetics is not sufficient to account for the night blindness. Recently, single rod recording from animal models of RP showed rods under degeneration remain saturated for shorter periods than normal rods; i.e. are less able to sustain the rod photoresponse. Using paired-flash ERG, here we determine whether rod phototransduction inactivation parameters might also be abnormal in patients with RP. Inactivation parameters were derived from 13 subjects with normal vision, 16 patients with adRP, and 16 patients with autosomal recessive/isolate (rec/iso) RP. The adRP cases included 9 patients with rhodopsin mutations and 7 patients with peripherin/RDS mutations. The inactivation phase was derived using a double-flash paradigm, with a test flash of 2.7 log scot td-s followed at varying intervals by a 4.2 log scot td-s probe flash. Derived rod photoresponses to this just-saturating test flash in normal subjects exhibit a critical time to the initiation of recovery (T sat) of 525 ± 90 (SD) ms. The values of T sat were 336 ± 104 (SD) ms in patients with adRP ( P < 0.001) and 271 ± 45 (SD) ms ( P < 0.001) in patients with rec/iso RP. When T sat values were categorized by mutations, the values were 294 ± 91 (SD) ms ( P < 0.001) for rhodopsin mutations, and 389 ± 100 (SD) ms ( p = 0.01) for peripherin/RDS mutations. Overall, T sat in patients with RP was significantly correlated with the amplitude of ISCEV standard rod response ( r = 0.56; P < 0.001) and the gain of the activation phase of phototransduction ( r = 0.6, P < 0.001). T sat may be a useful marker for therapeutic efficacy in future clinical trials in RP.

Effects of anti-factor VIII inhibitor antibodies on factor VIIa/tissue factor-catalysed activation and inactivation of factor VIII

Factor (F)VIIa/tissue factor (TF) rapidly activates FVIII activity by proteolysis at Arg372 and Arg740, and subsequently inactivates FVIIIa activity by proteolysis at Arg336, although this activation is weaker than that by thrombin. The effects of anti-FVIII inhibitor antibodies on these reactions remain unknown, however. In this study, 13 of anti-FVIII inhibitor antibodies recognising the A2 or C2 domain were prepared. None of them, irrespective of epitope specificity, significantly affected FVIIa/TF-catalysed FVIII activation in one-stage clotting assays. Anti-A2 and anti-C2 type 2 antibodies had little effect on the inactivation phase. Anti-C2 type 1 antibodies, however, modulated inactivation by 40-60% of that seen with control IgG, suggesting that the activity of FVIIIa generated by FVIIa/TF persisted in the presence of this specific type of inhibitor. SDS-PAGE analysis demonstrated that all antibodies had little effect on FVIIa/TF-catalyzed proteolysis at Arg372 and Arg740. Anti-C2 type 1, however, significantly delayed cleavage at Arg336 in dose- dependent manners. Neither anti-A2 nor anti-C2 type 2 affected this reaction, and the findings were consistent with the results of the functional assays. In addition, anti-C2 monoclonal antibodies with type 1 and 2 demonstrated similar patterns of reaction as the anti-C2 polyclonal antibodies in FVIIa/TF-mediated FVIII mechanisms. We demonstrated that FVIIa/TF activated FVIII even in the presence of anti-FVIII antibodies, but inactivation patterns appeared to depend on inhibitor type. It could be important to determine the characteristic of these inhibitor antibodies for prediction of their effects on FVIIa-related FVIII reactions, and the results could have significant therapeutic implications.

Cross‐Linking of Lipases Adsorbed on Hydrophobic Supports: Highly Selective Hydrolysis of Fish Oil Catalyzed by RML

AbstractOrganic cosolvents may improve the properties of lipases (e.g., selectivity); however, organic cosolvents also promote the desorption of the enzyme from its hydrophobic supports. In this study, adsorbed lipase molecules were cross‐linked with polyfunctional polymers, such as aldehyde‐dextrans, to prevent this desorption. The desorption of adsorbed lipases was greatly reduced by optimizing the polymer size, polymer/lipase ratio, and cross‐linking time. More than 95% of cross‐linked, immobilized Rhizomucor miehei lipase (RML) remained adsorbed on the support after washing with cosolvents or detergents. This new, immobilized RML preparation mediated the hydrolysis of sardine oil in the presence of organic cosolvents. The presence of cosolvents promoted small losses of hydrolytic activity. Interestingly, however, 50% 2‐propanol also promoted increased selectivity in the release of eicosapentaenoic acid (EPA) in relation to docosahexaenoic acid (DHA). An EPA/DHA ratio of 4:1 in the absence of 2‐propanol was increased to a ratio of 22:1 in the presence of 2‐propanol. The new RML derivatives were relatively stable under the selected reaction conditions. Their overall half‐life was 100 h, but, in a second inactivation phase (below 60% of remaining activity), it took 600 h to reach 30% of their remaining activity.

Phenotypes and peripheral mechanisms underlying inflammatory pain-related behaviors induced by BmK I, a modulator of sodium channels

The integrated mechanisms of dynamic signaling of sodium channels involved in clinical pain are still not yet clear. In this study, a new rat inflammatory pain model was developed by using the unilateral intraplantar injection of BmK I, a receptor site 3-specific modulator of sodium channels from the venom of scorpion Buthus martensi Karsch (BmK). It was found that BmK I could induce several kinds of inflammatory pain-related behaviors including spontaneous pain companied with unique episodic paroxysms, primary thermal hypersensitivity, and mirror-image mechanical hypersensitivity with different time course of development, which could be suppressed by morphine, indomethacin, or bupivacaine to a different extent. The dramatic attenuation by pretreatment with resiniferatoxin (RTX), an ultrapotent analog of capsaicin, on BmK I-induced pain-related behaviors, paw edema, and spinal L4–L5 c-Fos expression demonstrated that capsaicin-sensitive primary afferent neurons played important roles in pain induced by BmK I. Furthermore, the electrophysiological recordings showed that BmK I persistently increased whole-cell and tetrodotoxin-resistant (TTX-R) peak sodium currents and significantly delayed the inactivation phase of whole-cell sodium currents but could not enhance capsaicin-evoked inward currents, in acute isolated small dorsal root ganglion neurons of rat. The results strongly suggested that the dynamic modulation of BmK I on sodium channels located in peripheral primary afferent neurons, especially in capsaicin-sensitive neurons, mediated pain sensation. Thus, BmK I may be a valuable pharmacological tool to understand the sodium channel-involved pain mechanisms.

Escherichia coli population heterogeneity: Subpopulation dynamics at super-optimal temperatures

In the past years, we explored the dynamics of Escherichia coli K12 at super-optimal temperatures under static and dynamic temperature conditions ( Van Derlinden et al. (2008b, 2009, 2010). Disturbed sigmoid growth curves, i.e., a sequence of growth, inactivation and re-growth, were observed, especially close to the maximum growth temperature. Based on the limited set of experiments (i.e., 2 static temperatures and 2 dynamic temperature profiles), the irregular growth curves were explained by postulating the co-existence of two subpopulations: a more resistant, growing population and a temperature sensitive, inactivating population. In this study, the dynamics of the two subpopulations are studied rigorously at 11 constant temperature levels in the region between 45 °C and 46 °C, with at least five repetitions per temperature. At all temperatures, the total population follows a sequence of growth, inactivation and re-growth. The sequence of different stages in the growth curves can be explained by the two subpopulations. The first growth phase and the inactivation phase reflect the presence of the sensitive subpopulation. Hereafter, the population’s dynamics are dominated by the growth of the resistant subpopulation. Generally, cell counts are characterized by a large variability. The dynamics of the two subpopulations are carefully analyzed using a heterogeneous subpopulation type model to study the relation between the kinetic parameters of the two subpopulations and temperature, and to evaluate if the fraction d of resistant cells varies with temperature. Results indicate that the growth rate of the sensitive subpopulation decreases with increasing temperature within the range of 45–46 °C. Furthermore, results point in the direction that the duration of this initial growth phase is approximately constant, i.e., around 2 h. Possibly, the stress resistance of the cells decreases after a certain period because the metabolism is fully adapted to exponential growth. Also, the growth rate of the resistant subpopulation decreases with increasing temperature. Due to the extreme variability in the cell density data, derivation of accurate relations was not possible. From the heterogeneous model implementations, given the experimental set-up, both a constant d value and a temperature dependent d value seem plausible.

Diet-induced alterations in hepatic progesterone (P4) catabolic enzyme activity and P4 clearance rate in lactating dairy cows

Elevated rates of steroid clearance may lead to lower reproductive success in several mammalian species. Cytochrome P450 (EC 1.14.14.1) and aldo-keto reductases (AKR; EC 1.1.1.145-151) are involved in the first phase of steroid inactivation, before second phase conjugation and excretion of the steroid metabolite. The current objectives were to determine liver blood flow (LBF), hepatic enzyme activity, and metabolic clearance rate (MCR) of progesterone (P(4)) in dairy cows consuming isoenergetic and isonitrogenous diets formulated to cause divergent insulin secretion. Insulin concentrations increased by 22% in cows fed the high cornstarch diet, and both cytochrome P450 2C and cytochrome P450 3A activities were decreased (P<0.05) by approximately 50%, while AKR1C tended (P<0.10) to be lower in cows fed the high cornstarch diet. LBF was similar between the two diets (1891+/-91 l/h). MCR of P(4) tended (P<0.10) to be lower in cows fed the high cornstarch diet (25+/-5 l/hxBW(0.75)) versus the high fiber diet (40+/-6 l/hxBW(0.75)). The half-life of P(4) was increased (P<0.05) in cows fed the high cornstarch diet (73+/-10 min) versus the high fiber diet (24+/-10 min). In summary, cows with elevated insulin concentrations and lower enzyme activity showed a decrease in P(4) clearance without any changes in LBF. This dietary relationship with hepatic enzyme activity may explain some of the observed alterations in steroid profiles during the estrous cycle or gestation of the high producing dairy cow.

Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels

The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.

Studies on Thermodynamics and Kinetics of Thermo- Inactivation of Some Quality-Related Enzymes in White Yam (Dioscorea rotundata)

The effect of heat treatment on the activities of three quality related enzymes:- peroxidase (POD), polyphenol oxidase (PPO), and lipoxygenase (LOX), from edible white yam (Dioscorea rotundata) was studied over a temperature range of 50 to 80°C using mathematical analysis of the kinetic and thermodynamic parameters for the thermoinactivation of the enzymes. Denaturation of these enzymes, measured by loss in activity, could be described by a simple first-order reaction that was resolved into biphasic inactivation curves. This indicates the existence of two isoforms of different thermal stabilities with k-values between 0.032 and 0.525. D-values decreased with increasing temperature, indicating faster inactivation of the enzymes at higher temperatures. Results suggested that peroxidase is relatively more thermostable than polyphenol oxidase and lipoxygenase with a Z-value of 4.11, Ea of 2510kJ mol-1 for the first phase of the biphasic inactivation reaction. The Gibbs free energy (ΔG), values range from -552.95 to 279.01kJ/mol for the three enzymes. The results indicate that the oxidation reactions were: (1) not spontaneous (ΔG > 0) for peroxidase at low temperatures, (2) spontaneous (ΔG 0) for lipoxygenase and (4) reversible (ΔS < 0) for all the three enzymes at all temperature. The high z-value obtained for peroxidase in the first phase of inactivation indicates that a high amount of energy was required to initiate its denaturation, and supports why it is used as a marker for inactivation of quality-related enzymes.

Ca2+‐dependent components of inactivation of unitary cardiac L‐type Ca2+ channels

A Ca(2+) ion-dependent inactivation (CDI) of L-type Ca(2+) channels (LCC) is vital in limiting and shaping local Ca(2+) ion signalling in a variety of excitable cell types. However, under physiological conditions the unitary LCC properties that underlie macroscopic inactivation are unclear. Towards this end, we have probed the gating kinetics of individual cardiac LCCs recorded with a physiological Ca(2+) ion concentration (2 mM) permeating the channel, and in the absence of channel agonists. Upon depolarization the ensemble-averaged LCC current decayed with a fast and a slow exponential component. We analysed the unitary behaviour responsible for this biphasic decay by means of a novel kinetic dissection of LCC gating parameters. We found that inactivation was caused by a rapid decrease in the frequency of LCC reopening, and a slower decline in mean open time of the LCC. In contrast, with barium ions permeating the channel ensemble-averaged currents displayed only a single, slow exponential decay and little time dependence of the LCC open time. Our results demonstrate that the fast and slow phases of macroscopic inactivation reflect the distinct time courses for the decline in the frequency of LCC reopening and the open dwell time, both of which are modulated by Ca(2+) influx. Analysis of the evolution of CDI in individual LCC episodes was employed to examine the stochastic nature of the underlying molecular switch, and revealed that influx on the order of a thousand Ca(2+) ions may be sufficient to trigger CDI. This is the first study to characterize both the unitary kinetics and the stoichiometry of CDI of LCCs with a physiological Ca(2+) concentration. These novel findings may provide a basis for understanding the mechanisms regulating unitary LCC gating, which is a pivotal element in the local control of Ca(2+)-dependent signalling processes.

Modeling the inactivation of Salmonella typhimurium by dense phase carbon dioxide in carrot juice

The inactivation of Salmonella typhimurium inoculated into acidified carrot juice subjected to dense phase carbon dioxide (DPCD) was investigated. The pressures in the study were 10, 20 and 30 MPa, the temperatures were 32, 37 and 42 °C, and the treatment time was 5–90 min. The inactivation effect of DPCD was enhanced by increasing pressure and temperature. The sigmoid inactivation curves were characterized with the lag phase, exponential inactivation phase, and resistant phase. The inactivation curves were fitted to the modified Gompertz equation and the modified Logistic equation, the modified Gompertz equation was superior since its lowest residual sum of squares (RSS) was lower although there was no significant difference of goodness-of-fit between both models as indicated by F-test. The λ (the duration of the lag phase) and t 4-D (the time necessary to achieve 4-log cycles reduction) decreased with increasing pressure or temperature. The k dm (the maximum specific value of the inactivation rate, min −1) increased with increasing temperatures, and decreased with increasing pressures. The activation energy ( Ea) and the activation volume ( Va) necessary for inactivating S. typhimurium by DPCD were 19.06–29.39 kJ mol −1 and 18.89–58.27 cm 3 mol −1.

Characterization ofBacillus subtilisspore inactivation in low-pressure, low-temperature gas plasma sterilization processes

To identify structural components of Bacillus subtilis spores serving as targets for sterilization with microwave induced low-pressure, low-temperature nitrogen-oxygen plasma. The inactivation of spores followed a biphasic kinetics consisting of a log-linear phase with rapid inactivation followed by a slow inactivation phase. In the course of plasma treatment, damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild-type strain showed the highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid-soluble proteins (Delta sspA Delta sspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV-C radiation is the most effective inactivation agent in the plasma, whereby the splB and Delta cotE mutant spores were equally and slightly less sensitive, respectively, than the wild-type spores. Finally, the extent of damages in the spore DNA determined by quantitative PCR correlated with the spore inactivation. Spore inactivation was efficiently mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. The results provide new evidence to the understanding of plasma sterilization processes. This knowledge supports the identification of useful parameters for novel plasma sterilization equipment to control process safety.

Kinetics of adenovirus type 2 inactivation with free chlorine

The objective of this study was to elucidate the effects of pH, temperature, and other relevant water quality parameters on the kinetics of adenovirus serotype 2 inactivation with free chlorine. Over a pH range of 6.5–10, a temperature range of 1–30 °C, and in a variety of water types, free chlorine was highly effective against adenovirus type 2. Its disinfection efficacy decreased with increasing pH and decreasing temperature, yet was unaffected by hardness and buffering species. Under the most challenging conditions investigated in this study (pH 10, 1 °C), a four-log reduction of adenovirus viability would be achieved at a CT value of 2.6 mg Cl 2 min/L. The inactivation kinetics was characterized by three phases of inactivation under most conditions. The first phase resulted from a reaction involving primarily the hypochlorous acid species and was characterized by rapid inactivation of viruses to a limit that increased with decreasing pH and increasing temperature. After reaching this limit, adenovirus exhibited two subsequent phases of inactivation at lesser rates that were not affected by temperature or pH. As with the first phase of kinetics, a limit of inactivation was approached in the second phase that decreased with increasing pH, and after which the kinetics was characterized by a third and final phase. An inactivation model consistent with these observations was found to provide adequate representation for the free chlorine inactivation of adenovirus serotype 2 as well as that reported in the literature for other adenovirus serotypes.

Modulation of L-type Ca2+ channel current density and inactivation by β-adrenergic stimulation during murine cardiac embryogenesis

L-type Ca(2+) current (I (CaL)) is a key regulatory and functional element during early embryonic cardiomyogenesis. As the embryonic heart underlies hormonal modulation, e.g. catecholamines, we aimed at studying effects of beta-adrenergic stimulation on I (CaL) densities and inactivation kinetics during murine heart development. I (CaL) was recorded applying the whole-cell patch-clamp technique in ventricular myocytes of early embryonic (EDS, E9.5-11.5) and late developmental, fetal (LDS, E16.5-18.5) stages as well as adult cardiomyocytes. To distinguish between Ca(2+)-(CDI) and voltage-dependent inactivation (VDI), Ca(2+) was replaced with Ba(2+) in the extracellular recording solution. The beta-adrenergic signaling pathway was simulated by applying isoproterenol (Iso). Basal current densities showed an increase of I (CaL) during development (EDS: -9.61 +/- 1.97 pA/pF, n = 9; LDS: -13.2 +/- 4.26 pA/pF, n = 12; adult: -16.1 +/- 4.63 pA/pF, n = 5). Iso (1 microM) enhanced I (CaL) density with low effects at EDS (17.1 +/- 3.58%, n = 8, P > 0.05) but strong effects at LDS (74.1 +/- 3.77%, n = 8, P < 0.01) and in adults (90.6 +/- 7.01%, n = 6, P < 0.001). The current availability was significantly higher at LDS as compared to EDS and elevated after application of Iso. In the presence of Ca(2+), the fast phase of I (CaL) inactivation (tau(f)) was significantly enhanced by Iso at LDS. The slow phase of inactivation (tau(s)) was unaltered at both developmental stages. However, VDI was significantly reduced under Iso in LDS and adult cardiomyocytes. These results imply that beta-adrenergic modulation becomes of importance especially during fetal heart development. CDI and VDI of I (CaL) are modulated by beta-adrenergic stimulation in fetal but not in early embryonic mouse cardiomyocytes. Furthermore our data suggest important changes of the L-type Ca(2+) channel protein, and/or maturation of the Ca(2+)-induced Ca(2+) release (CICR) machinery.

Inactivating potassium currents in apical and basal turn inner hair cells from guinea-pig cochlea

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. IHCs of guinea-pigs were separately isolated from the apical and basal turns and the tonotopic gradient of inactivation kinetics was investigated. TEA-sensitive potassium currents showed voltage-dependent time constant of the inactivation phase both in apical and basal IHCs, however, the degree of inactivation (compared to the ratio between the steady-state current and initial peak current) was voltage-independent. Inactivation time constant was faster in basal IHCs than in apical IHCs and the degree of inactivation was greater in basal IHCs than in apical IHCs, suggesting that inactivation was more predominant in basal IHCs than in apical IHCs.

Role of N-Terminal Domain and Accessory Subunits in Controlling Deactivation-Inactivation Coupling of Kv4.2 Channels

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2Δ2–10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2Δ2–10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.