Depolarizing Pulse Research Articles

Slc25a3-dependent copper transport controls flickering-induced Opa1 processing for mitochondrial safeguard

Following the Goldilocks principle, mitochondria size must be "just right." Mitochondria balance division and fusion to avoid becoming too big or too small. Defects in this balance produce dysfunctional mitochondria in human diseases. Mitochondrial safeguard (MitoSafe) is a defense mechanism that protects mitochondria against extreme enlarging by suppressing fusion in mammalian cells. In MitoSafe, hyperfused mitochondria elicit flickering-short pulses of mitochondrial depolarization. Flickering activates an inner membrane protease, Oma1, which in turn proteolytically inactivates a mitochondrial fusion protein, Opa1. The mechanisms underlying flickering are unknown. Using a live-imaging screen, we identified Slc25a3 (a mitochondrial carrier transporting phosphate and copper) as necessary for flickering and Opa1 cleavage. Remarkably, copper, but not phosphate, is critical for flickering. Furthermore, we found that two copper-containing mitochondrial enzymes, superoxide dismutase 1 and cytochrome c oxidase, regulate flickering. Our data identify an unforeseen mechanism linking copper, redox homeostasis, and membrane flickering in mitochondrial defense against deleterious fusion.

Effects of Kv1.3 knockout on pyramidal neuron excitability and synaptic plasticity in piriform cortex of mice.

Kv1.3 belongs to the voltage-gated potassium (Kv) channel family, which is widely expressed in the central nervous system and associated with a variety of neuropsychiatric disorders. Kv1.3 is highly expressed in the olfactory bulb and piriform cortex and involved in the process of odor perception and nutrient metabolism in animals. Previous studies have explored the function of Kv1.3 in olfactory bulb, while the role of Kv1.3 in piriform cortex was less known. In this study, we investigated the neuronal changes of piriform cortex and feeding behavior after smell stimulation, thus revealing a link between the olfactory sensation and body weight in Kv1.3 KO mice. Coronal slices including the anterior piriform cortex were prepared, whole-cell recording and Ca2+ imaging of pyramidal neurons were conducted. We showed that the firing frequency evoked by depolarization pulses and Ca2+ influx evoked by high K+ solution were significantly increased in pyramidal neurons of Kv1.3 knockout (KO) mice compared to WT mice. Western blotting and immunofluorescence analyses revealed that the downstream signaling molecules CaMKII and PKCα were activated in piriform cortex of Kv1.3 KO mice. Pyramidal neurons in Kv1.3 KO mice exhibited significantly reduced paired-pulse ratio and increased presynaptic Cav2.1 expression, proving that the presynaptic vesicle release might be elevated by Ca2+ influx. Using Golgi staining, we found significantly increased dendritic spine density of pyramidal neurons in Kv1.3 KO mice, supporting the stronger postsynaptic responses in these neurons. In olfactory recognition and feeding behavior tests, we showed that Kv1.3 conditional knockout or cannula injection of 5-(4-phenoxybutoxy) psoralen, a Kv1.3 channel blocker, in piriform cortex both elevated the olfactory recognition index and altered the feeding behavior in mice. In summary, Kv1.3 is a key molecule in regulating neuronal activity of the piriform cortex, which may lay a foundation for the treatment of diseases related to piriform cortex and olfactory detection.

Cucurbit[6]uril Hyperpolarized Chemical Exchange Saturation Transfer Pulse Sequence Parameter Optimization and Detectability Limit Assessment at 3.0T.

Molecular imaging is the future of personalized medicine; however, it requires effective contrast agents. Hyperpolarized chemical exchange saturation transfer (HyperCEST) can boost the signal of Hyperpolarized 129Xe MRI and render it a molecular imaging modality of high efficiency. Cucurbit[6]uril (CB6) has been successfully employed in vivo as a contrast agent for HyperCEST MRI, however its performance in a clinical MRI scanner has yet to be optimized. In this study, MRI pulse sequence parameter optimization was first performed in CB6 solutions in phosphate-buffered saline (PBS), and subsequently in whole sterile citrated bovine blood. The performance of four different depolarization pulse shapes (sinusoidal, 3-lobe sinc (3LS), rectangular (block), and hyperbolic secant (hypsec) was optimized. The detectability limits of CB6 in a clinical 3.0T MRI scanner was assessed using the optimized pulse sequences. The 3LS depolarization pulses performed best, and demonstrated 24% depletion in a 25µM solution of CB6 in PBS. It performed similarly in blood. The CB6 detectability limit was found to be 100µM in citrated bovine blood with a correspondent HyperCEST depletion of 30% ± 9%. For the first time, the HP 129Xe HyperCEST effect was observed in red blood cells (RBC) and had a similar strength as HyperCEST in plasma.

Characterization in Potent Modulation on Voltage-Gated Na+ Current Exerted by Deltamethrin, a Pyrethroid Insecticide

Deltamethrin (DLT) is a type-II pyrethroid ester insecticide used in agricultural and domestic applications as well as in public health. However, transmembrane ionic channels perturbed by this compound remain largely unclear, although the agent is thought to alter the gating characteristics of voltage-gated Na+ (NaV) channel current. In this study, we reappraised whether and how it and other related compounds can make any further modifications on voltage-gated Na+ current (INa) in pituitary tumor (GH3) cells. Cell exposure to DLT produced a differential and dose-dependent stimulation of peak (transient, INa(T)) or sustained (late, INa(L)) INa; consequently, the EC50 value required for DLT-stimulated INa(T) or INa(L) was determined to be 11.2 or 2.5 μM, respectively. However, neither the fast nor slow component in the inactivation time constant of INa(T) activated by short depolarizing pulse was changed with the DLT presence; conversely, tefluthrin (Tef), a type-I pyrethroid insecticide, can accentuate INa with a slowing in inactivation time course of the current. The INa(L) augmented by DLT was attenuated by further application of either dapagliflozin (Dapa) or amiloride, but not by chlorotoxin. During pulse train (PT) stimulation, with the Tef or DLT presence, the cumulative inhibition of INa(T) became slowed; moreover, following PT stimuli, a large tail current with a slowly recovering process was observed. Alternatively, during rapid depolarizing pulse, the amplitude of INa(L) and tail INa (INa(Tail)) for each depolarizing pulse became progressively increased by adding DLT, not by Tef. The recovery time constant following PT stimulation with continued presence of Tef or DLT was shortened by further addition of Dapa. The voltage-dependent hysteresis (Hys(V)) of persistent INa was differentially augmented by Tef or DLT. Taken together, the magnitude, gating, frequency dependence, as well as Hys(V) behavior of INa exerted by the presence of DLT or Tef might exert a synergistic impact on varying functional activities of excitable cells in culture or in vivo.

State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from Halichondria okadai.

Voltage-gated sodium channels (Nav) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge Halichondria okadai; however, toxic substances that modulate Nav are yet to be identified. This study aimed to identify Nav inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Nav subtype Nav1.7, using whole-cell patch-clamp recordings. We successfully isolated arachidonic acid (AA, 1) from the hexane extract of H. okadai, and then the fatty acid-mediated modulation of Nav in N2A was investigated in detail for the first time. Octanoic acid (2), palmitic acid (3), and oleic acid (4) showed no inhibitory activity at 100 μM, whereas AA (1), dihomo-γ-linolenic acid (DGLA, 5), and eicosapentaenoic acid (EPA, 6) showed IC50 values of 6.1 ± 2.0, 58 ± 19, and 25 ± 4.0 μM, respectively (N = 4, mean ± SEM). Structure and activity relationships were investigated for the first time using two ω-3 polyunsaturated fatty acids (PUFAs), EPA (6) and eicosatetraenoic acid (ETA, 7), and two ω-6 PUFAs, AA (1) and DGLA (5), to determine their effects on a resting state, activated state, and inactivated state. Steady-state analysis showed that the half inactivation potential was largely hyperpolarized by 10 μM AA (1), while 50 μM DGLA (5), 50 μM EPA (6), and 10 μM ETA (7) led to a slight change. The percentages of the resting state block were 24 ± 1, 22 ± 1, 34 ± 4, and 38 ± 9% in the presence of AA (1), DGLA (5), EPA (6), and ETA (7), respectively, with EPA (6) and ETA (7) exhibiting a greater inhibition than both AA (1) and DGLA (5), and their inhibitions did not increase in the following depolarization pulses. None of the compounds exhibited the use-dependent block. The half recovery times from the inactivated state for the control, AA (1), DGLA (5), EPA (6), and ETA (7) were 7.67 ± 0.33, 34.3 ± 1.10, 15.5 ± 1.10, 10.7 ± 0.31, and 3.59 ± 0.18 ms, respectively, with AA (1) exhibiting a distinctively large effect. Overall, distributed binding to the resting and the inactivated states of Nav would be significant for the inhibition of Nav, which presumably depends on the active structure of each PUFA.

The effect of fast charging and equalization on the reliability and cycle life of the lead acid batteries

Three-wheeled e-rickshaws driven by lead-acid batteries are a common means of transport in Asian countries due to their low cost, and durability. Conventional charging is time-consuming and lacks proper controls for the termination of the charge. Though fast charging can significantly reduce the charging time to about one-third or less, it is prone to temperature rise, excessive gassing, and reduction in the useful life of the battery. A new fast-charging procedure along with periodic equalizing is proposed in this paper. A multistage constant current fast charger with depolarization pulse was used to charge a 48 V e-rickshaw battery pack in 3 h 37 min from 20 % to 80 % state of charge. The effect of the said fast charging procedure on the coulombic efficiency, end voltage pattern, capacity degradation, reliability, and useful life of the lead-acid batteries is investigated. Experimental results for 150 charging-discharging cycles show a temperature rise up to 5–6 °C, average coulombic efficiency of 93 %, and a maximum top-of-charge voltage of 2.6 Volts Per Cell (VPC). Attainment of such a low-temperature rise coupled with high coulombic efficiency during fast charge is the most competitive result reported for commercial batteries.A regression model and best-fit probability distribution (Weibull 3-parameter distribution) are used to estimate the degradation rate, and Cycle-To-Failure (CTF). The expected life of the batteries subjected to such a fast charging and equalizing charge is predicted to be 1296 cycles, which is about 2 times the current life of the battery. The proposed fast charger along with periodic equalizing is, therefore, a promising and sustainable option for charging lead-acid batteries.

Smart Pacemaker: A Review.

Since the first pacemaker was implanted, nearly 60 years have passed. Since then, pacemaker technology has made major advancements that have increased both its safety and effectiveness in treating people with bradyarrhythmias. The repeated stimulation of cells in specialized "pacemaker" regions of the mammalian heart and the transmission of stimulus via the ventricles serve as evidence that the electrical function of the mammalian heart is necessary for a regular mechanical (pump) role. The development of action potentials in individual cardiac cells is linked to myocardial electrical activity and the heart's regular cooperative electrical functioning. A container or pulse initiator that houses the battery and electronics, as well as lines that connect to the myocardium to deliver a depolarizing pulse and detect intrinsic cardiac stimulation, are all parts of a pacemaker. Defibrillators could be used with artificial hearts that have electrical pacemakers integrated into them in order to treat arrhythmia, heart failure, and cardiac arrest. Modern pacemakers have units for supporting patients with other disorders like "heart failure," which happens when the heart does not pump as forcefully as it should. While many pacemakers are effective in treating different types of arrhythmias (irregular heartbeats), they also have units for treating them.

Activation of Voltage-Gated Na+ Current by GV-58, a Known Activator of CaV Channels.

GV-58 ((2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol) is recognized to be an activator of N- and P/Q-type Ca2+ currents. However, its modulatory actions on other types of ionic currents in electrically excitable cells remain largely unanswered. This study was undertaken to explore the possible modifications caused by GV-58 in ionic currents (e.g., voltage-gated Na+ current [INa], A-type K+ current [IK(A)], and erg-mediated K+ current [IK(erg)]) identified from pituitary GH3 lactotrophs. GH3 cell exposure to GV-58 enhanced the transient and late components of INa with varying potencies; consequently, the EC50 values of GV-58 required for its differential increase in peak and late INa in GH3 cells were estimated to be 8.9 and 2.6 μM, respectively. The INa in response to brief depolarizing pulse was respectively stimulated or suppressed by GV-58 or tetrodotoxin, but it failed to be altered by ω-conotoxin MVIID. Cell exposure to this compound increased the recovery of INa inactivation evoked by two-pulse protocol based on a geometrics progression; however, in its presence, there was a slowing in the inactivation rate of current decay evoked by a train of depolarizing pulses. The existence of GV-58 also resulted in an increase in the amplitude of ramp-induced resurgent and window INa. The presence of this compound inhibited IK(A) magnitude, accompanied by a shortening in inactivation time course of the current; however, it mildly decreased IK(erg). Under current-clamp conditions, GV-58 increased the frequency of spontaneous action potentials in GH3 cells. Moreover, in NSC-34 motor neuron-like cells, the presence of GV-58 not only raised INa amplitude but also reduced current inactivation. Taken together, the overall work provides a noticeable yet unidentified finding which implies that, in addition to its agonistic effect on Ca2+ currents, GV-58 may concertedly modify the amplitude and gating kinetics of INa in electrically excitable cells, hence modifiying functional activities in these cells.

Quantification of the calcium signaling deficit in muscles devoid of triadin.

Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice. We find higher resting cytosolic and lower SR-luminal [Ca2+], 40% lower calsequestrin expression, and more CaV1.1, RyR1 and SERCA1. Despite the increased CaV1.1, the mobile intramembrane charge was reduced by ~20% in Triadin-null fibers. The initial peak of calcium release flux by pulse depolarization was minimally altered in the null fibers (revealing an increase in peak calcium permeability). The “hump” phase that followed, attributable to calcium detaching from calsequestrin, was 25% lower, a smaller change than expected from the reduced calsequestrin content and calcium saturation. The exponential decay rate of calcium transients was 25% higher, consistent with the higher SERCA1 content. Recovery of calcium flux after a depleting depolarization was faster in triadin-null myofibers, consistent with the increased uptake rate and lower SR calsequestrin content. In sum, the triadin knockout determines an increased RyR1 channel openness, which depletes the SR, a substantial loss of calsequestrin and gains in other couplon proteins. Powerful functional compensations ensue: activation of SOCE that increases [Ca2+]cyto; increased SERCA1 activity, which limits the decrease in [Ca2+]SR and a restoration of SR calcium storage of unknown substrate. Together, they effectively limit the functional loss in skeletal muscles.

The effects of an initial depolarization pulse on dissolved phase hyperpolarized 129Xe brain MRI

To evaluate the effect of an initial 90° depolarization RF pulse on the dissolved-phase hyperpolarized (HP) xenon-129 (129 Xe) brain imaging and to compare the SNR variability of HP 129 Xe images acquired without an initial depolarization RF pulse to those following the initial depolarization pulse. Five cognitive normal healthy volunteers were imaged using a Philips Achieva 3.0T MRI scanner during a single breath-hold following inhalation of 1 L of HP 129 Xe. Each participant underwent six HP 129 Xe scans. Three scans were performed using conventional single-slice projection HP 129 Xe brain imaging, and the other three scans were performed using the HP 129 Xe time-of-flight imaging with an initial rectangular depolarization pulse. Although the utilization of an initial depolarization results in the reduction of the mean image SNR, the presence of an initial depolarization RF pulse reduces the SNR variability of the HP 129 Xe brain image by a factor of 2.26. The highest SNR variability was observed from the posterior brain region, where the anterior region possessed the lower level of signal variability. An initial 90° depolarization RF pulse, applied prior to the HP 129 Xe image acquisition, reduced the HP 129 Xe signal variability more than two times between the different breath-hold images.

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering‐induced Oma1 activation

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin-related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin-related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1-knockout cells. Re-activation of Opa1 and Mitofusin 1 in Drp1-knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.

State-dependent Photocrosslinking at the BK Channel Intersubunit Interface

BK channels are high conductance potassium channels synergistically activated by voltage and Ca2+. Their molecular architecture consists on homotetramers of α-subunits containing a membrane-spanning domain and a cytosolic structure including two RCK-like domains (RCK1 and RCK2). Each RCK contains distinct high affinity Ca2+-binding sites that have been proposed to mediate activation of the BK channel by Ca2+ (Horrigan and Aldrich, 2002; Sweet and Cox., 2008). The binding site in RCK2, known as the Ca2+-bowl, is located very close to the intersubunit interface in the gating-ring. The cryo-EM structure of the full-length BK channel from Aplysia revealed that Ca2+ in this site is additionally coordinated by the side chain of residue N438 from the RCK1 region of the adjacent subunit (human N449; Tao et al., 2017). Functional data with the BKα-N449A mutant from Li et al., 2018; Vouga and Rothberg, 2017, highlight the importance of this residue in the Ca2+ activation mechanism. To further study the role of N449 in BK channel activation by Ca2+ and voltage we took advantage of the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine (BzF), which was incorporated at that specific site in the human BK channel. BK-N449BzF constructs showed functional characteristics undistinguishable from wild-type BK channels. UV-induced BzF photocrosslinking led to a state-dependent reduction of the maximal outward currents. Photocrosslinking of closed channels (UV application during hyperpolarizing pulses) rendered faster and complete current decrease. Conversely, when the UV pulse was applied during the depolarization pulses (open channels), the current decay was incomplete and developed more slowly. Biochemical experiments confirmed intersubunit crosslinking. Further experiments with BK channels including mutations to inactivate the Ca2+ bowl are currently underway to unveil specific structural prerequisites for this intersubunit interaction. Overall, these results implicate a movement at this subunit interface in BK channel activation.

Temperature Dependence of the Biophysical Mechanisms Underlying the Inhibition and Enhancement Effect of Amiodarone on hERG Channels.

hERG K+ channel is important for controlling the duration of cardiac action potentials. Amiodarone (AMD), a widely prescribed class III antiarrhythmic, could inhibit hERG currents with relatively few tachyarrhythmic adverse events. We use injected Xenopus oocyte with two-electrode voltage clamp techniques to characterize the action of AMD on hERG channels. We found that AMD binds to the resting hERG channel with an apparent dissociation constant of ∼1.4 μM, and inhibits hERG currents at mild and strong depolarization pulses by slowing activation and enhancing inactivation, respectively, at 22°C. The activation kinetics of hERG channel activation are much faster, but inactivation kinetics are slower at 37°C. AMD accordingly has a 15% to 20% weaker and stronger inhibitory effect at mild and strong depolarization (e.g., -60 and +30 mV, 0.3-second pulse), respectively. In the meanwhile, the resurgent hERG tail currents are dose-dependently inhibited by AMD without altering the kinetics of current decay at both 22°C and 37°C, indicating facilitation of recovery from inactivation via the silent route. Most importantly, AMD no longer inhibits but enhances hERG currents at a mild pulse shortly after a prepulse at 37°C, but not so much at 22°C. We conclude that AMD is an effective hERG channel-gating modifier capable of lengthening the plateau phase of cardiac action potential (without increasing the chance of afterdepolarization). AMD, however, should be used with caution in hypothermia or the other scenarios that slow hERG channel activation. SIGNIFICANCE STATEMENT: It is known that amiodarone (AMD) acts on hERG K+ channels to treat cardiac arrhythmias with relatively little arrhythmogenicity. We found that AMD enhances hERG channel inactivation but slows activation as well as recovery from inactivation, and thus has a differential inhibition and enhancement effect on hERG currents at different phases of membrane voltage changes, especially at 37°C, but not so much at 22°C. AMD is therefore a relatively ideal agent against tachyarrhythmia at 37°C, but should be more cautiously used at lower temperatures or relevant pathophysiological/pharmacological scenarios associated with slower hERG channel activation because of the increased chances of adverse events.

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca2+ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca2+ channels, in particular with the CaV2.2 α1-subunit and describe its modulatory effects on the current through CaV2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with CaV2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein βγ subunits on CaV2 channels. The impact of Grina/TMBIM3 and G-protein βγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.

Dehydroepiandrosterone inhibits ICa,L and its window current in voltage-dependent and -independent mechanisms in arterial smooth muscle cells.

Dehydroepiandrosterone (DHEA) is an adrenal steroid hormone, which has the highest serum concentration among steroid hormones with DHEA sulfate (DHEAS). DHEA possesses an inhibitory action on glucose-6-phosphate dehydrogenase (G6PD), the first pentose-phosphate pathway enzyme that reduces NADP+ to NADPH. DHEA induced relaxation of high K+-induced contraction in rat arterial strips, whereas DHEAS barely induced it. We studied the effects of DHEA on L-type Ca2+ current ( ICa,L) of A7r5 arterial smooth muscle cells and compared the mechanism of inhibition with that produced by the 6-aminonicotinamide (6-AN) competitive inhibitor of G6PD. DHEA moderately inhibited ICa,L that was elicited from a holding potential (HP) of -80 mV [voltage-independent inhibition (VIDI)] and accelerated decay of ICa,L during the depolarization pulse [voltage-dependent inhibition (VDI)]. DHEA-induced VDI decreased peak ICa,L at depolarized HPs. By applying repetitive depolarization pulses from multiple HPs, novel HP-dependent steady-state inactivation curves ( f∞-HP) were constructed. DHEA shifted f∞-HP to the left and inhibited the window current, which was recorded at depolarized HPs and obtained as a product of current-voltage relationship and f∞-HP. The IC50 value of ICa,L inhibition was much higher than serum concentration. DHEA-induced VDI was downregulated by the dialysis of guanosine 5'- O-(2-thiodiphosphate), which shifted f∞-voltage to the right before the application of DHEA. 6-AN gradually and irreversibly inhibited ICa,L by VIDI, suggesting that the inhibition of G6PD is involved in DHEA-induced VIDI. In 6-AN-pretreated cells, DHEA induced additional inhibition by increasing VIDI and generating VDI. The inhibition of G6PD underlies DHEA-induced VIDI, and DHEA additionally induces VDI as described for Ca2+ channel blockers. NEW & NOTEWORTHY Dehydroepiandrosterone, the most abundantly released adrenal steroid hormone with dehydroepiandrosterone sulfate, inhibited L-type Ca2+ current and its window current in aortic smooth muscle cells. The IC50 value of inhibition decreased with the depolarization of holding potential to 15 µM at -20 mV. The inhibition occurred in a voltage-dependent manner as described for Ca2+ channel blockers and in a voltage-independent manner because of the inhibition of glucose-6-phosphate dehydrogenase.

Regulation of Action Potential Frequency and Amplitude by T-type Ca2+ Channel During Spontaneous Synchronous Activity of Hippocampal Neurons

In this paper, the changes in the frequency and amplitude of action potentials (AP) were investigated depending on the depolarization caused by the Ca2+ channels activity during the spontaneous synchronous activity (SSA) of hippocampal neurons in culture. It is known that the pacemaker neuronal electrical activity can be both tonic and bursting. Using the image analysis to measure [Са2+]i and patch-clamp to register the membrane potential we show that depolarization caused by the GABA(A) receptor inhibitor results in a pattern of SSA in which tonic APs frequency of 2−3 Hz are generated by a neuron without any changes in cytosolic free Ca2+ concentration, ([Ca2+]i). The tonic mode is interrupted by bursts activity, which is accompanied by slow depolarization and calcium pulses. The inhibitor of T-type calcium channels, ML218, suppresses this process. The frequency and amplitude of the AP are regulated by slow depolarization pulses as follows: on the depolarization front, the APs frequency increases. At the same time, the amplitude decreases due to Na+ channels inactivation. The higher is the depolarization rate, the higher is the APs frequency. If slow depolarization amplitude exceeds Na+ channels reactivation potential, the neuronal impulse activity stops. As the [Ca2+]i increases and Ca2+-dependent K+ channels are activated, depolarization amplitude decreases slowly, and Na+ channels are reactivated, which leads to a gradual increase in the amplitude of APs against the background of depolarization decrease. The frequency of APs on the backside of depolarization pulse slows down to 3–4 Hz due to the occurrence of the faster Ca2+-potential oscillations (micro bursts of AP). Under these conditions, only one AP can be generated due to rapid depolarization at the leading edge. After that, APs generation is suppressed due to Na+ channel inactivation. The frequency of APs in this case coincides with the Ca2+ channel activation frequency (3−4 Hz). The burst firing is terminated due to [Ca2+]i increase, Ca2+-dependent K+ channels activation, and voltage-gated Ca2+ channels (VGCC) inactivation. As a result, the membrane is hyperpolarized even more (10 mV lower than the critical potential), that suppress AP generation, activate HCN-like channels and reactivate Na+ and VGCC. The activity of HCN-like channels increases, the membrane slowly depolarizes and reaches the threshold potential. The generation of tonic APs begins, and then, the Ca2+ channels opens, and Ca2+ potential and Ca2+ signal induced again. Thus, the Ca2+-channels, is determining the pulse of slow depolarization, control the frequency and the amplitude of APs during SSA, regulating the activation and inactivation conditions of Na+ channels. The T-type channels inhibitors reduce the duration of burst firing and Ca2+ impulse in neurons in vitro, stimulating their survival during hyperexcitation and ischemia. Thus, reducing the Ca2+ pulse duration caused by the inhibitors of T-type Ca2+-channels, can be one of the reasons for the known neuroprotective effect of these compounds. Keywords: spontaneous synchronous activity of neurons, calcium signal, T-type calcium channels, voltage-gated calcium channels, action potential, genesis of bursting activity, action potential bursts, depolarization shift, critical potential

The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression

Elephantopus scaber Linn and its major bioactive component, deoxyelephantopin are known for their medicinal properties and are often reported to have various cytotoxic and antitumor activities. This plant is widely used as folk medicine for a plethora of indications although its safety profile remains unknown. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. The hERG potassium channel is an important antitarget in cardiotoxicity evaluation. This study investigated the effects of deoxyelephantopin on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells. The hERG tail currents following depolarization pulses were insignificantly affected by deoxyelephantopin in the transfected cell line. Current reduction was less than 40% as compared with baseline at the highest concentration of 50 μM. The results were consistent with the molecular docking simulation and hERG surface protein expression. Interestingly, it does not affect the hERG expression at both transcriptional and translational level at most concentrations, although higher concentration at 10 μM caused protein accumulation. In conclusion, deoxyelephantopin is unlikely a clinically significant hERG channel and Ikr blocker.

Activity-induced spontaneous spikes in GABAergic neurons suppress seizure discharges: an implication of computational modeling.

BackgroundEpilepsy, a prevalent neurological disorder, appears self-termination. The endogenous mechanism for seizure self-termination remains to be addressed in order to develop new strategies for epilepsy treatment. We aim to examine the role of activity-induced spontaneous spikes at GABAergic neurons as an endogenous mechanism in the seizure self-termination.Methods and ResultsNeuronal spikes were induced by depolarization pulses at cortical GABAergic neurons from temporal lobe epilepsy patients and mice, in which some of these neurons fired activity-induced spontaneous spikes. Neural networks including excitatory and inhibitory neurons were computationally constructed, and their functional properties were based on our studies from whole-cell recordings. With the changes in the portion and excitability of inhibitory neurons that generated activity-induced spontaneous spike, the efficacies to suppress synchronous seizure activity were analyzed, such as its onset time, decay slope and spike frequency. The increases in the proportion and excitability of inhibitory neurons that generated activity-induced spontaneous spikes effectively suppressed seizure activity in neural networks. These factors synergistically strengthened the efficacy of seizure activity suppression.ConclusionOur study supports a notion that activity-induced spontaneous spikes in GABAergic neurons may be an endogenous mechanism for seizure self-termination. A potential therapeutic strategy for epilepsy is to upregulate the cortical inhibitory neurons that generate activity-induced spontaneous spikes.

Temperature Changes of Lead Acid Battery Cell with Pulse Charging in a Flooded State

The paper describes temperature changes of lead acid battery cell in the course of discharging and pulse charging in a flooded construction. There was found effect of different pulse charging with and without depolarization pulse on increase of cell temperature compared with constant current charging mode.

Mitragynine and its potential blocking effects on specific cardiac potassium channels

Mitragyna speciosa Korth is known for its euphoric properties and is frequently used for recreational purposes. Several poisoning and fatal cases involving mitragynine have been reported but the underlying causes remain unclear. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. On the other hand, IK1, a Kir current mediated by Kir2.1 channel and IKACh, a receptor-activated Kir current mediated by GIRK channel are also known to be important in maintaining the cardiac function. This study investigated the effects of mitragynine on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells and Xenopus oocytes. The effects on Kir2.1 and GIRK channels currents were also determined in the oocytes. The hERG tail currents following depolarization pulses were inhibited by mitragynine with an IC50 value of 1.62μM and 1.15μM in the transfected cell line and Xenopus oocytes, respectively. The S6 point mutations of Y652A and F656A attenuated the inhibitor effects of mitragynine, indicating that mitragynine interacts with these high affinity drug-binding sites in the hERG channel pore cavity which was consistent with the molecular docking simulation. Interestingly, mitragynine does not affect the hERG expression at the transcriptional level but inhibits the protein expression. Mitragynine is also found to inhibit IKACh current with an IC50 value of 3.32μM but has no significant effects on IK1. Blocking of both hERG and GIRK channels may cause additive cardiotoxicity risks.