Use-dependent Manner Research Articles

The calcium sensor synaptotagmin 7 is required for synaptic facilitation.

It has been known for over 70 years that synaptic strength is dynamically regulated in a use-dependent manner1. At synapses with a low initial release probability, closely spaced presynaptic action potentials can result in facilitation, a short-term form of enhancement where each subsequent action potential evokes greater neurotransmitter release2. Facilitation can enhance neurotransmitter release manyfold and profoundly influence information transfer across synapses3, but the underlying mechanism remains a mystery. Among the proposed mechanisms is that a specialized calcium sensor for facilitation transiently increases the probability of release2,4 and is distinct from the fast sensors that mediate rapid neurotransmitter release. Yet such a sensor has never been identified, and its very existence has been disputed5,6. Here we show that synaptotagmin 7 (syt7) is a calcium sensor that is required for facilitation at multiple central synapses. In syt7 knockout mice, facilitation is eliminated even though the initial probability of release and presynaptic residual calcium signals are unaltered. Expression of wild-type syt7 in presynaptic neurons restored facilitation, whereas expression of a mutated syt7 with a calcium-insensitive C2A domain did not. By revealing the role of syt7 in synaptic facilitation, these results resolve a longstanding debate about a widespread form of short-term plasticity, and will enable future studies that may lead to a deeper understanding of the functional importance of facilitation.

Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K+ (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) value of 0.81μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77±0.04μM−1s−1 and 2.55±1.50s−1, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition.

Fluvoxamine by itself has potential to directly induce long QT syndrome at supra-therapeutic concentrations.

Fluvoxamine is one of the typical selective serotonin-reuptake inhibitors. While its combined use with QT-prolonging drugs has been contraindicated because of the increase in plasma concentrations of such drugs, information is still limited whether fluvoxamine by itself may directly prolong the QT interval. We examined electropharmacological effects of fluvoxamine together with its pharmacokinetic profile by using the halothane-anesthetized dogs (n = 4). Fluvoxamine was intravenously administered in three escalating doses of 0.1, 1 and 10 mg/kg over 10 min with a pause of 20 min between the doses. The low dose provided therapeutic plasma drug concentration, whereas the middle and high doses attained approximately 10 and 100 times of the therapeutic ones, respectively. Supra-therapeutic concentration of fluvoxamine exerted the negative chronotropic, inotropic and hypotensive effects; and suppressed the atrioventricular nodal and intraventricular conductions, indicating inhibitory actions on Ca2+ and Na+ channels, whereas it delayed the repolarization in a reverse use-dependent manner, reflecting characteristics of rapidly activating delayed rectifier K+ current channel-blocking property. Fluvoxamine prolonged the terminal repolarization phase at 100 times higher concentration than the therapeutic, indicating its proarrhythmic potential. Thus, fluvoxamine by itself has potential to directly induce long QT syndrome at supra-therapeutic concentrations.

Application of an in vitro model for early detection of cardiac sodium channel blocking liabilities

Off-target inhibition of the human cardiac sodium channel (hNav1.5) can prolong the QRS interval and has been associated with increased mortality in patients with underlying cardiovascular diseases. Given the cardiac safety implications for patients and the costs of late stage drug development, detection and mitigation of hNav1.5 liabilities early in drug discovery and development is critically important. Cardiac Na channel activities are typically tested using in vitro methods i.e. patch clamp in transfected cells, followed by in vivo evaluation. Two classes of compounds showed dose-dependent QRS prolongation in the dog telemetry studies despite little in vitro activity on Nav1.5 (Compound 1, up to 100 μM) or weak activity (Compound 2) with IC50 of 46 μM) in a patch clamp assay. Putatively a more predictive in vitro assay for detecting Nav1.5 inhibition, a rabbit ventricular wedge (RVW) was used given its sensitivity for detecting Nav1.5 activity. Methods and Results: Jacket telemetry was used to record surface ECG’s in beagle dogs (n=3). The wedge preparations (n=4, male, New Zealand White rabbits) were perfused with Tyrode’s solution via the coronary artery in a tissue bath. The dog findings demonstrated a dosedependent increase in QRS intervals after administration of compound 1 or 2. The RVW indicated both compound 1 and 2 produced a concentration-dependent increase in QRS duration and loss of excitation at 100 μM for compound 2, indicative of inward Na current (INa) inhibition. Both compounds prolonged QRS duration in a usedependent manner. Conclusions: The RVW reliably detects cardiac Na channel blockade in a use-dependent manner, but that had tested negative on Nav1.5 patch clamp tests. Thus, the rabbit wedge assay can be used to detect Nav1.5 blockade, and is a valuable addition to be incorporated in early testing.

Azithromycin Can Prolong QT Interval and Suppress Ventricular Contraction, but Will Not Induce Torsade de Pointes.

Azithromycin has been reported to increase the risk of death from cardiovascular causes among patients with high baseline risk. Since the information is still limited to bridge the gap between electrophysiological properties of azithromycin in vitro and cardiac death in patients, we initially assessed its electropharmacological effects in doses of 3 and 30 mg/kg, i.v., with the halothane-anesthetized dogs (n = 4). The low dose provided 5.2 times higher than the therapeutic concentration, whereas the high dose attained 17.0 times higher. The high dose delayed the ventricular repolarization in a reverse use-dependent manner, reflecting blockade of the rapid component of delayed rectifier K(+) current, and the potency was relatively weak; namely, maximum change in QTc was +20 ms (+5.6%). The high dose also induced the negative inotropic effect possibly through Ca(2+) channel-independent pathway. In order to clarify proarrhythmic risk, 30 mg/kg, i.v., of azithromycin was examined with the chronic atrioventricular block dogs (n = 4). Azithromycin neither induced torsade de pointes nor affected beat-to-beat variability of repolarization. Thus, azithromycin can be considered to lack proarrhythmic potential, but caution has to be paid on its use for patients with left ventricular dysfunction.

Function of inhibitory micronetworks is spared by Na+ channel-acting anticonvulsant drugs.

The mechanisms of action of many CNS drugs have been studied extensively on the level of their target proteins, but the effects of these compounds on the level of complex CNS networks that are composed of different types of excitatory and inhibitory neurons are not well understood. Many currently used anticonvulsant drugs are known to exert potent use-dependent blocking effects on voltage-gated Na(+) channels, which are thought to underlie the inhibition of pathological high-frequency firing. However, some GABAergic inhibitory neurons are capable of firing at very high rates, suggesting that these anticonvulsants should cause impaired GABAergic inhibition. We have, therefore, studied the effects of anticonvulsant drugs acting via use-dependent block of voltage-gated Na(+) channels on GABAergic inhibitory micronetworks in the rodent hippocampus. We find that firing of pyramidal neurons is reliably inhibited in a use-dependent manner by the prototypical Na(+) channel blocker carbamazepine. In contrast, a combination of intrinsic and synaptic properties renders synaptically driven firing of interneurons essentially insensitive to this anticonvulsant. In addition, a combination of voltage imaging and electrophysiological experiments reveal that GABAergic feedforward and feedback inhibition is unaffected by carbamazepine and additional commonly used Na(+) channel-acting anticonvulsants, both in control and epileptic animals. Moreover, inhibition in control and epileptic rats recruited by in vivo activity patterns was similarly unaffected. These results suggest that sparing of inhibition is an important principle underlying the powerful reduction of CNS excitability exerted by anticonvulsant drugs.

Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain

Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using β-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at −40mV, peaked at −26mV, and half-inactivated at −46.7mV. The persistent sodium current (INaP) activated at −51mV, peaked at −23mV, and half-inactivated at −30.7mV. Tetrodotoxin (TTX; 1μM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd2+ (50μM) potently blocked INaP with little effect on INaf. Riluzole (>20μM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at −30mV, peaked at −11.3mV, and half-inactivated at −34mV. The Ca2+ channel blocker verapamil (100μM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca2+-activated K+ (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca2+ or adding verapamil or nifedipine, and increased by adding the Ca2+ agonist Bay K8644, indicating that Ca2+ entry via the L-type Ca2+ channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100μM riluzole in the bath solution. These membrane oscillations disappeared with 1μM TTX, 50μM Cd2+, replacement of external Na+ with choline, and blockage of Na+-activated K+ current (IKNa) with 50μM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in Ca2+-free medium or in the presence of verapamil were eliminated by blocking INaP with 50μM Cd2+. Taken together, these results indicate that INaP and IKNa participate in the generation of membrane oscillations and that INaP additionally participates in the generation of plateau potentials and initiation of spontaneous action potentials. ICa, through L-type Ca2+ channels, was also found to play a role in the rapid membrane repolarization of action potentials by functional coupling with BK channels.

Early and moderate sensory stimulation exerts a protective effect on perilesion representations of somatosensory cortex after focal ischemic damage.

Previous studies have shown that intensive training within an early critical time window after focal cortical ischemia increases the area of damaged tissue and is detrimental to behavioral recovery. We postulated that moderate stimulation initiated soon after the lesion could have protective effects on peri-infarct cortical somatotopic representations. Therefore, we have assessed the effects of mild cutaneous stimulation delivered in an attention-demanding behavioral context on the functional organization of the perilesion somatosensory cortex using high-density electrophysiological mapping. We compared the effects of 6-day training initiated on the 3rd day postlesion (early training; ET) to those of same-duration training started on the 8th day (delayed training; DT). Our findings confirm previous work showing that the absence of training aggravates representational loss in the perilesion zone. In addition, ET was found to be sufficient to limit expansion of the ischemic lesion and reduce tissue loss, and substantially maintain the neuronal responsiveness to tactile stimulation, thereby preserving somatotopic map arrangement in the peri-infarct cortical territories. By contrast, DT did not prevent tissue loss and only partially reinstated lost representations in a use-dependent manner within the spared peri-infarct cortical area. This study differentiates the effects of early versus delayed training on perilesion tissue and cortical map reorganization, and underscores the neuroprotective influence of mild rehabilitative stimulation on neuronal response properties in the peri-infarct cortex during an early critical period.

QPEEG analysis of the effects of sodium valproate on adult Chinese patients with generalized tonic-clonic seizures.

Objectives EEG effects of the sustained-release form of sodium valproate (SR-VPA) are unknown, although it is widely used in Chinese patients with generalized tonicclonic seizures (GTCS). Methods Fourteen newly diagnosed, untreated GTCS patients were recruited and treated with SR-VPA. Waking EEG was recorded and analyzed by way of quantitative pharmaco-electroencephalogram (QPEEG) analysis during the three-month follow-up. Results There was a statistically significant decrease in the absolute power of the delta band (P < 0.05), theta band (P < 0.03) and partial alpha-1 band (p < 0.05) with treatment compared to before treatment, while there was no significantly different absolute power between one-month and three-months after treatment. There was a strong correlation between the decrease in absolute power and the degree of the initial abnormality in all frequency bands. Two of 14 patients experienced seizures during the second month after initiation of SR-VPA therapy. Conclusions SR-VPA selectively decreased the activity of the abnormal EEG synchronization in a use-dependent manner. The reduced theta, delta, and partial alpha-1 absolute power may reflect or confirm the efficacy of SR-VPA on patients with GTCS.

Control of thin filament lengths by sarcomeric tropomodulin isoforms: insights from mouse models (1102.2)

The uniform lengths of skeletal muscle thin filaments are controlled by actin subunit exchange at pointed ends (P‐ends). Each P‐end is capped by two sarcomeric tropomodulin (Tmod) molecules, with Tmod1 and Tmod4 isoforms present in a 1:9 ratio. In Tmod1‐null sarcomeres, P‐ends are capped by Tmod3 (normally a sarcoplasmic reticulum‐associated Tmod isoform) and Tmod4, preserving normal thin filament lengths. In Tmod4‐null sarcomeres, P‐ends are capped solely by Tmod1, also preserving normal lengths. The mild phenotypes of Tmod1‐null and Tmod4‐null muscle highlight skeletal muscle’s ability to deploy compensatory mechanisms to specify correct thin filament lengths in the absence of one sarcomeric Tmod isoform during myofibril assembly and muscle development. To discern the effect of sarcomeric Tmod depletion from mature muscle, we measured thin filament lengths in the mdx and mdx/mTR mouse models of Duchenne muscular dystrophy, in which Tmods are calpain‐proteolyzed in a muscle use‐dependent manner. Proteolysis of Tmod1 and/or Tmod4 in mdx and mdx/mTR muscles results in 10‐12% increases in thin filament lengths, due to addition of actin subunits onto P‐ends. Collectively, these results show that Tmod1 and Tmod4 are individually dispensable for initial specification of muscle‐specific thin filament lengths, but important for maintenance of correct thin filament lengths in mature contracting myofibrils.Grant Funding Source: Supported by NIH and MDA.

The calmodulin inhibitor and antipsychotic drug trifluoperazine inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effect of the calmodulin inhibitor and antipsychotic drug trifluoperazine on voltage-dependent K+ (Kv) channels. Kv currents were recorded by whole-cell configuration of patch clamp in freshly isolated rabbit coronary arterial smooth muscle cells. The amplitudes of Kv currents were reduced by trifluoperazine in a concentration-dependent manner, with an apparent IC50 value of 1.58±0.48μM. The rate constants of association and dissociation by trifluoperazine were 3.73±0.33μM−1s−1 and 5.84±1.41s−1, respectively. Application of trifluoperazine caused a positive shift in the activation curve but had no significant effect on the inactivation curve. Furthermore, trifluoperazine provoked use-dependent inhibition of the Kv current under train pulses (1 or 2Hz). These findings suggest that trifluoperazine interacts with Kv current in a closed state and inhibits Kv current in the open state in a time- and use-dependent manner, regardless of its function as a calmodulin inhibitor and antipsychotic drug.

Blockade of Nav1.8 Currents in Nociceptive Trigeminal Neurons Contributes to Anti-trigeminovascular Nociceptive Effect of Amitriptyline

Amitriptyline (AMI), a tricyclic antidepressant, has been widely used to prevent migraine attacks and alleviate other various chronic pain, but the underlying mechanism remains unclear. Accumulated evidence suggests that the efficacy of AMI is related to the blockade of voltage-gated sodium channels. The aim of the present study was to investigate the effect of AMI on Na(v)1.8 currents in nociceptive trigeminal neurons and trigeminovascular nociception induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus (SSS) in rats, as in the animal model of vascular headaches such as migraines. Using a whole-cell voltage recording technique, we showed that Na(v)1.8 currents were blocked by AMI in a concentration-dependent manner, with an IC50 value of 6.82 μM in acute isolated trigeminal ganglion neurons of the rats. AMI caused a hyperpolarizing shift in the voltage-dependent activation and steady-state inactivation and significantly blocked in a use-dependent manner and slowed the recovery from the inactivation of Na(v)1.8 currents. In addition, the systemic administration of AMI and A-803467 (a selective Na(v)1.8 channel blocker) potently alleviated the nociceptive behaviors (head flicks and grooming) induced by the electrical stimulation of the dura mater surrounding the SSS. Taken together, our data suggest that Na(v)1.8 currents in nociceptive trigeminal neurons are blocked by AMI through modulating the activation and inactivation kinetics, which may contribute to anti-nociceptive effect of AMI in animal models of migraines.

Apamin Does Not Block Major Cardiac Depolarization and Repolarization Currents

Background Recent findings indicate that small-conductance Ca 2+ -activated K + (SK) channels are abundantly present in atrial tissues and are upregulated in the diseased ventricles. Almost all studies used apamin, which is a specific SK channel blocker in neurons that does not block any other neural type of ion channels. However, the specificity of apamin in blocking SK currents in cardiac tissues remains unclear because apamin has been reported to block fetal L-type Ca 2+ current in embryonic chick heart. The purpose of the present study was to test the hypothesis that apamin does not block any major cardiac type ion currents. Methods We studied human embryonic kidney (HEK293) cells that either stably expressed human voltage-gated Na + currents or were transiently transfected to express major human cardiac type K + and Ca 2+ currents. Whole-cell patch-clamp techniques were used to determine ionic current densities before and after apamin administration. Results Ca 2+ currents ( CACNA1C+CACNAB2B ) in 4 cells (–17 ± 4.9 pA/pF) were not affected by apamin (500 nM) (–17 ± 5.9 pA/pF, P = NS) but were reduced by nifedipine (2 µM) to –5 ± 4.0 pA/pF ( P = .008). Na + currents (SCN5A) in 9 cells (–231 ± 96 pA/pF) were not affected by apamin (500 nM) (–232 ± 107 pA/pF, P = NS) but were reduced by flecainide (100 µM) in a use-dependent manner to 71 ± 51 pA/pF ( P = Ks (KCNQ1+KCNE1) (25 ± 9 pA/pF) was not blocked by apamin (500 nM) (n = 8, 22 ± 9 pA/pF, P = NS) but was blocked by chromanol 293B (50 µM) to 5 ± 2 pA/pF (n = 4, P = .01). I Kr (KCNH2+KCNE2) (27 ± 8 pA/pF) was not blocked by apamin (500 nM) (n = 6, 26 ± 6 pA/pF, P = NS) but was blocked by E4031 (100 nM) to 13 ± 4 pA/pF (n = 6, P K1 (KCNJ2) (–45 ± 14 pA/pF) was not blocked by apamin (500 nM) (n = 7, –46 ± 17 pA/pF, P = NS) but was blocked by CsCl (5 mM) to –16 ± 5 pA/pF (n = 7, P = .004). I to (KCND3) (526 ± 115 pA/pF) was not blocked by apamin (500 nM) (n = 2, 516 ± 110 pA/pF, P = NS) but was blocked by 4-aminopyridine (10 mM) to 235 pA/pF (n = 1). Conclusions Apamin does not block human Na + current, L-type Ca 2+ current, or other major K + currents in HEK293 cells. These findings indicate that apamin is a specific SK current blocker in human cardiac tissues.

Proton-dependent inhibition of the cardiac sodium channel Nav1.5 by ranolazine

Ranolazine is clinically approved for treatment of angina pectoris and is a potential candidate for antiarrhythmic, antiepileptic, and analgesic applications. These therapeutic effects of ranolazine hinge on its ability to inhibit persistent or late Na+ currents in a variety of voltage-gated sodium channels. Extracellular acidosis, typical of ischemic events, may alter the efficiency of drug/channel interactions. In this study, we examined pH modulation of ranolazine's interaction with the cardiac sodium channel, Nav1.5. We performed whole-cell path clamp experiments at extracellular pH 7.4 and 6.0 on Nav1.5 transiently expressed in HEK293 cell line. Consistent with previous studies, we found that ranolazine induced a stable conformational state in the cardiac sodium channel with onset/recovery kinetics and voltage-dependence resembling intrinsic slow inactivation. This interaction diminished the availability of the channels in a voltage- and use-dependent manner. Low extracellular pH impaired inactivation states leading to an increase in late Na+ currents. Ranolazine interaction with the channel was also slowed 4–5 fold. However, ranolazine restored the voltage-dependent steady-state availability profile, thereby reducing window/persistent currents at pH 6.0 in a manner comparable to pH 7.4. These results suggest that ranolazine is effective at therapeutically relevant concentrations (10 μM), in acidic extracellular pH, where it compensates for impaired native slow inactivation.

The inhibitory effect of curcumin on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of curcumin, the principal active compound of turmeric, on voltage-dependent K(+) (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Curcumin reduced the Kv current in a dose-dependent manner with an apparent K(d) value of 1.07 ± 0.03 μM. Although curcumin did not alter the kinetics of Kv current activation, it predominantly accelerated the decay rate of channel inactivation. The association and dissociation rate constants of curcumin were 1.35 ± 0.05 μM(-1)s(-1) and 1.47 ± 0.17s(-1), respectively. Curcumin did not alter the steady-state activation or inactivation curves. Application of train pulses (1 or 2 Hz) increased curcumin-induced blockade of the Kv current, and the recovery time constant also increased in the presence of curcumin suggesting, that the inhibitory action of Kv currents by curcumin was use-dependent. From these results, we concluded that curcumin inhibited vascular Kv current in a state-, time-, and use-dependent manner.

Effect of Daurisoline on hERG Channel Electrophysiological Function and Protein Expression

Daurisoline (1) is a bis-benzylisoquinoline alkaloid isolated from the rhizomes of Menispermum dauricum. The antiarrhythmic effect of 1 has been demonstrated in different experimental animals. In previous studies, daurisoline (1) prolonged action potential duration (APD) in a normal use-dependent manner. However, the electrophysiological mechanisms for 1-induced prolongation of APD have not been documented. In the present study, the direct effect of 1 was investigated on the hERG current and the expression of mRNA and protein in human embryonic kidney 293 (HEK293) cells stably expressing the hERG channel. It was shown that 1 inhibits hERG current in a concentration- and voltage-dependent manner. In the presence of 10 μM 1, steady-state inactivation of V(1/2) was shifted negatively by 15.9 mV, and 1 accelerated the onset of inactivation. Blockade of hERG channels was dependent on channel opening. The expression and function of hERG were unchanged by 1 at 1 and 10 μM, while hERG expression and the hERG current were decreased significantly by 1 at 30 μM. These results indicate that 1, at concentrations below 30 μM, exerts a blocking effect on hERG, but does not affect the expression and function of the hERG channel. This may explain the relatively lower risk of long QT syndrome after long-term usage.

Central Mechanisms of Menthol-Induced Analgesia

Menthol is one of the most commonly used chemicals in our daily life, not only because of its fresh flavor and cooling feeling but also because of its medical benefit. Previous studies have suggested that menthol produces analgesic action in acute and neuropathic pain through peripheral mechanisms. However, the central actions and mechanisms of menthol remain unclear. Here, we report that menthol has direct effects on the spinal cord. Menthol decreased both ipsilateral and contralateral pain hypersensitivity induced by complete Freund's adjuvant in a dose-dependent manner. Menthol also reduced both first and second phases of formalin-induced spontaneous nocifensive behavior. We then identified the potential central mechanisms underlying the analgesic effect of menthol. In cultured dorsal horn neurons, menthol induced inward and outward currents in a dose-dependent manner. The menthol-activated current was mediated by Cl(-) and blocked by bicuculline, suggesting that menthol activates γ-aminobutyric acid type A receptors. In addition, menthol blocked voltage-gated sodium channels and voltage-gated calcium channels in a voltage-, state-, and use-dependent manner. Furthermore, menthol reduced repetitive firing and action potential amplitude, decreased neuronal excitability, and blocked spontaneous synaptic transmission of cultured superficial dorsal horn neurons. Liquid chromatography/tandem mass spectrometry analysis of brain menthol levels indicated that menthol was rapidly concentrated in the brain when administered systemically. Our results indicate that menthol produces its central analgesic action on inflammatory pain probably via the blockage of voltage-gated Na(+) and Ca(2+) channels. These data provide molecular and cellular mechanisms by which menthol decreases neuronal excitability, therefore contributing to menthol-induced central analgesia.

Cloning and functional analysis of P2X1b, a new variant in rat optic nerve that regulates the P2X1 receptor in a use-dependent manner.

P2X receptors are trimeric, ATP-gated cation channels. In mammals seven P2X subtypes have been reported (P2X1-P2X7), as well as several variants generated by alternative splicing. Variants confer to the homomeric or heteromeric channels distinct functional and/or pharmacological properties. Molecular biology, biochemical, and functional analysis by electrophysiological methods were used to identify and study a new variant of the P2X1 receptor named P2X1b. This new variant, identified in rat optic nerve, was also expressed in other tissues. P2X1b receptors lack amino acids 182 to 208 of native P2X1, a region that includes residues that are highly conserved among distinct P2X receptors. When expressed in Xenopus oocytes, P2X1b was not functional as a homomer; however, when co-expressed with P2X1, it downregulated the electrical response generated by ATP compared with that of oocytes expressing P2X1 alone, and it seemed to form heteromeric channels with a modestly enhanced ATP potency. A decrease in responses to ATP in oocytes co-expressing different ratios of P2X1b to P2X1 was completely eliminated by overnight pretreatment with apyrase. Thus, it is suggested that P2X1b regulates, through a use-dependent mechanism, the availability, in the plasma membrane, of receptor channels that can be operated by ATP.

Scorpion β-toxin interference with NaV channel voltage sensor gives rise to excitatory and depressant modes

Scorpion β toxins, peptides of ∼70 residues, specifically target voltage-gated sodium (NaV) channels to cause use-dependent subthreshold channel openings via a voltage–sensor trapping mechanism. This excitatory action is often overlaid by a not yet understood depressant mode in which NaV channel activity is inhibited. Here, we analyzed these two modes of gating modification by β-toxin Tz1 from Tityus zulianus on heterologously expressed NaV1.4 and NaV1.5 channels using the whole cell patch-clamp method. Tz1 facilitated the opening of NaV1.4 in a use-dependent manner and inhibited channel opening with a reversed use dependence. In contrast, the opening of NaV1.5 was exclusively inhibited without noticeable use dependence. Using chimeras of NaV1.4 and NaV1.5 channels, we demonstrated that gating modification by Tz1 depends on the specific structure of the voltage sensor in domain 2. Although residue G658 in NaV1.4 promotes the use-dependent transitions between Tz1 modification phenotypes, the equivalent residue in NaV1.5, N803, abolishes them. Gating charge neutralizations in the NaV1.4 domain 2 voltage sensor identified arginine residues at positions 663 and 669 as crucial for the outward and inward movement of this sensor, respectively. Our data support a model in which Tz1 can stabilize two conformations of the domain 2 voltage sensor: a preactivated outward position leading to NaV channels that open at subthreshold potentials, and a deactivated inward position preventing channels from opening. The results are best explained by a two-state voltage–sensor trapping model in that bound scorpion β toxin slows the activation as well as the deactivation kinetics of the voltage sensor in domain 2.