Half-activation Potential Research Articles

The voltage-dependent structural transitions governing the opening of the human t-type Cav3.1 channel: Insights from voltage-clamp fluorometry.

Low-voltage-activated CaV3.1 (T-type) Ca2+ channels contribute to neuronal burst-mode firing and heart pacemaker activity. Similar to other voltage-gated channels, the four Voltage-Sensing Domains (VSDs) are expected to activate before the pore opens. However, previous studies attempting to characterize the CaV3.1 voltage-sensing apparatus in channels blocked by La3+ found that most of the gating charge moves after the channel has opened. To shed light on the molecular mechanisms underlying the unique voltage-dependent gating of CaV3.1, we expressed the human CaV3.1 pore-forming subunit α1G in Xenopus oocytes and used voltage-clamp fluorometry (VCF) to optically track the structural rearrangements of the four VSDs. In conducting channels under physiologically-relevant conditions (2mM [Ca2+]out) we found that the activation curves, F(V), of all VSDs preceded the activation of the ionic conductance, G(V): the half-activation potentials of the four F(V) curves ranged between −61±2 mV and −42±1 mV, while G(V) was V1/2(1) = −36±2 mV; V1/2(2) = 6±3 mV. However, in channels blocked by 200 µM La3+ (as in gating current measurements), the voltage dependence of VSD-I, -III and -IV shifted towards depolarized potentials, to the right of the G(V) curve. Only VSD-II activation was not perturbed by La3+. These results suggest that VSD-I, -III and -IV are energetically coupled with the pore and likely responsible for the voltage-dependent activation. Accordingly, the ataxia-causing mutation R1715H (VSD-IV S4) caused a +20 mV shift in VSD-IV activation and a concomitant +10 mV shift in the G(V). Our findings demonstrate that under conducting conditions all VSDs of Cav3.1 operate in a canonical way: they activate before pore opening. We propose that the impaired activation of CaV3.1 carrying the ataxia-causing mutation is caused by the impaired activation of VSD-IV.

Dapagliflozin exhibits class I antiarrhythmic effects which suppress action potential formation in human atrial cardiomyocytes

Abstract Background Recently, inhibitors of sodium-glucose transporter 2 (SGLT2i) were shown to have tremendous cardioprotective effects in patients with type 2 diabetes mellitus (T2DM) as well as heart failure (HF) patients, regardless of their glycemic status. Among other beneficial effects on cardiovascular outcome an antiarrhythmic effect of SGLT2i was indicated. In patients with T2DM therapy with SGLT2i was associated with a significantly reduced risk of atrial arrhythmias and sudden cardiac death. As sodium-glucose transporter 2 (SGLT2) is not expressed within the heart underlying molecular modes of action remain unclear. Therefore, investigating possible antiarrhythmic mechanisms is crucial to determine whether SGLT2i might be beneficial for all patients suffering from arrhythmias, regardless of other comorbidities. Purpose To assess antiarrhythmic effects of SGLT2i on a molecular and cellular level. Therefore, direct electrophysiological effects of dapagliflozin – the most advanced SGLT2i in clinical trials – on action potential (AP) formation in atrial cardiomyocytes (CM) was investigated. Methods Effects of dapagliflozin on human NaV1.5, heterologously expressed in Chinese hamster ovary (CHO) cells, was investigated using the patch-clamp method. Further, effects on voltage-gated sodium channels (NaV) were also measured in human atrial CM. Consequences of dapagliflozin treatment on AP formation were studied on isolated human and porcine atrial CM using the patch-clamp technique. APs were elicited in current-clamp mode by injection of brief current pulses. Results Dapagliflozin (100 μM) significantly decreased peak sodium currents in human atrial CM as well as CHO cells, expressing human NaV1.5 channel subunits. Additionally, half-activation potential of voltage-gated sodium channels was significantly increased after administration of dapagliflozin. Furthermore, dapagliflozin (100 μM) suppressed AP formation in CM isolated from human and porcine left and right atrial tissue. In human CM action potential amplitude (APA) was significantly reduced by 34.3%, while APA inhibition by dapagliflozin averaged out at 30.7% for porcine CM. In porcine CM action potential duration at 50% (APD50) and 90% repolarization (APD90) were also significantly reduced. Conclusion Dapagliflozin suppresses AP formation in human and porcine atrial CM by inhibiting voltage-gated sodium currents. Therefore, we suppose that dapagliflozin exhibits class I antiarrhythmic effects. Funding Acknowledgement Type of funding sources: None.

FC025: Physiological Role of KV Channel in Ureter Smooth Muscle Cell Investigated Quantitatively by Electrophysiological Modeling

Abstract BACKGROUND AND AIMS Spontaneous peristaltic contraction disorder of the ureter smooth muscle (USM) is an important etiology of congenital giant ureter, congenital ureteropelvic junction deformity and non-obstructive hydronephrosis [1]. The USM cell action potential (AP) plays a key role in commencing USM contraction by allowing influx of Ca2+ from the extra cellular space [2]. Therefore, an in-depth quantitative investigation of the USM cell AP generation will assist in developing better pharmacological therapies for the USM abnormal contraction. The purpose of this in-silico study is to unfold the physiological role of Kv channel in modulating the USM AP generation. METHOD The model consists voltage- dependent Na+ currents (INa), hyperpolarization-activated cyclic nucleotide- gated current (Ih), voltage- dependent Ca2+ currents (ICa), Ca2+ dependent potassium channel (IBKCa) and voltage-dependent potassium channels (IKv) as the components of active electrophysiological characteristics [3]. All ion channels are represented by the Hodgkin–Huxley formalism. The maximum conductance of the is varied to investigate the physiological role of the Kv channel in modulating the USM AP generation. RESULTS The whole cell IKv current is generated by applying the voltage clamp protocol, where the membrane potential is stepped from a holding potential of –80 mV–60 mV for a duration of 500 ms. The half activation potential, half inactivation potential, activation slope factor and inactivation slope factors are: 43 mV, –76.1 mV, 15.4 mV and 6.5 mV, respectively, to fit the Boltzmann function equation. The model reveals that fast activation and inactivation properties are the essential feature of IKv for the USM cell. The resting membrane potential (RMP) is set at –50 mV. The AP was induced in the whole cell model by applying an external stimulus current (10–30 pA), as a brief square pulse of 500 ms duration. It is demonstrated that IKv opposes the Ca2+ current during the first 50 ms of the AP duration and then inactivated. It doesn't contribute to the repolarizing phase of the AP. CONCLUSION The plateau phase of the USM AP shows that the total AP duration much longer than that in other smooth muscle cells. It strongly supports the phenomenon is due to the activation of Ca2+-dependent K+ channel and inactivation of L-type Ca2+ channel. In ureter cells, the IKv does not contribute significantly to the repolarization of the AP, but it may regulate membrane excitability by opposing Ca2+ current activated around the threshold of the AP. Therefore, the agonist and antagonist of the IKv might be useful partially as new pharmacological targets for the USM abnormal contraction.

The Neuroprotective Effects of the CB2 Agonist GW842166x in the 6-OHDA Mouse Model of Parkinson's Disease.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder associated with dopamine neuron loss and motor dysfunction. Neuroprotective agents that prevent dopamine neuron death hold great promise for slowing the disease’s progression. The activation of cannabinoid (CB) receptors has shown neuroprotective effects in preclinical models of neurodegenerative disease, traumatic brain injury, and stroke, and may provide neuroprotection against PD. Here, we report that the selective CB2 agonist GW842166x exerted protective effects against the 6-hydroxydopamine (6-OHDA)-induced loss of dopamine neurons and its associated motor function deficits in mice, as shown by an improvement in balance beam walking, pole, grip strength, rotarod, and amphetamine-induced rotation tests. The neuroprotective effects of GW842166x were prevented by the CB2 receptor antagonist AM630, suggesting a CB2-dependent mechanism. To investigate potential mechanisms for the neuroprotective effects of GW842166x, we performed electrophysiological recordings from substantia nigra pars compacta (SNc) dopamine neurons in ex vivo midbrain slices prepared from drug-naïve mice. We found that the bath application of GW842166x led to a decrease in action potential firing, likely due to a decrease in hyperpolarization-activated currents (Ih) and a shift of the half-activation potential (V1/2) of Ih to a more hyperpolarized level. Taken together, the CB2 agonist GW842166x may reduce the vulnerability of dopamine neurons to 6-OHDA by decreasing the action potential firing of these neurons and the associated calcium load.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Modulation of human T-type calcium channels by synthetic cannabinoid receptor agonists in vitro

Background and purposeConsumption of Synthetic Cannabinoid Receptor agonists (SCRAs) is associated with severe adverse reactions including seizures, arrhythmias and death, but the molecular mechanisms surrounding SCRA toxicity are not yet established. These disease-like symptoms are also synonymous with altered T-type calcium channel activity which controls rhythmicity in the heart and brain. This study examined whether SCRAs alter T-type activity and whether this represents a possible mechanism of toxicity. Experimental approachFluorescence-based and electrophysiology assays were used to screen 16 structurally related synthetic cannabinoids for their ability to inhibit human T-type calcium channels expressed in HEK293 cells. The most potent compounds were then further examined using patch clamp electrophysiology. Key resultsMDMB-CHMICA and AMB-CHMINACA potently blocked Cav3.2 with IC50 values of 1.5 and 0.74 μM respectively. Current inhibition increased from 47 to 80% and 45–87% respectively when the channel was in slow-inactivated state. Both SCRAs had little effect on steady state inactivation, however MDMB-CHMICA significantly shifted the half activation potential by -7mV. Neither drug produced frequency dependent block, in contrast to the phytocannabinoid Δ9-THC. Conclusions and implicationsSCRAs are potent agonists of CB1 receptors and can be extremely toxic, but observed toxicity also resembles symptoms associated with altered Cav3.2 activity. Many SCRAs tested were potent modulators of Cav3.2, raising the possibility that SC toxicity may be due in part to Cav3.2 modulation. This potent T-type channel modulation suggests the possibility of SCRAs as a new drug class with potential to treat diseases associated with altered T-type channel activity.This article is part of the special issue on ‘Cannabinoids’.

Electrophysiological Identification and Activity Analyses of Plasma Membrane K+ Channels in Maize Guard Cells.

Stomatal movement, which plays an essential role in plant transpiration and photosynthesis, is controlled by ion channels that mediate K+ and anion fluxes across the plasma membrane (PM) of guard cells. These channels in dicots are accurately regulated by various physiological factors, such as pH, abscisic acid (ABA) and Ca2+; however, the data in monocots are limited. Here the whole-cell patch-clamping technique was applied to analyze the properties and regulations of PM K+ channels in maize guard cells. The results indicated that the hyperpolarization-activated inward-rectifying channels were highly K+-selective. These inward K+ (Kin) channels were sensitive to extracellular K+. Their slope factor (S) decreased when the apoplastic K+ concentration decline, causing a positive shift of the half-activation potential (V1/2). Their activities were promoted by apoplastic acidification but inhibited by apoplastic and cytosolic alkalization. Nevertheless, the outward K+ (Kout) channel activities were uniquely promoted by cytosolic alkalization. Both apoplastic and cytosolic ABA inhibited Kin channels independent of cytosolic Ca2+ ([Ca2+]cyt). And two Ca2+-dependent mechanisms with different Ca2+ affinities may mediate resting- and high-[Ca2+]cyt-induced inhibition on Kin channels, respectively. However, resting [Ca2+]cyt impaired the inhibition of Kin channels induced by apoplastic ABA, not cytosolic ABA. Furthermore, the result that high [Ca2+]cyt attenuated ABA-induced inhibition highlighted the importance of [Ca2+]cyt for Kin channel regulation. There may exist a Ca2+-dependent regulation of the Ca2+-independent ABA signaling pathways for Kin channel inhibition. These results provided an electrophysiological view of the multiple level regulations of PM K+ channel activities and kinetics in maize guard cells.

Ih contributes to increased motoneuron excitability in restless legs syndrome.

Restless legs patients complain about sensory and motor symptoms leading to sleep disturbances. Symptoms include painful sensations, an urge to move and involuntary leg movements. The responsible mechanisms of restless legs syndrome are still not known, although current studies indicate an increased neuronal network excitability. Reflex studies indicate the involvement of spinal structures. Peripheral mechanisms have not been investigated so far. In the present study, we provide evidence of increased hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated inward rectification in motor axons. The excitability of sensory axons was not changed. We conclude that, in restless legs syndrome, an increased HCN current in motoneurons may play a pathophysiological role, such that these channels could represent a valuable target for pharmaceutical intervention. Restless legs syndrome is a sensorimotor network disorder. So far, the responsible pathophysiological mechanisms are poorly understood. In the present study, we provide evidence that the excitability of peripheral motoneurons contributes to the pathophysiology of restless legs syndrome. In vivo excitability studies on motor and sensory axons of the median nerve were performed on patients with idiopathic restless legs syndrome (iRLS) who were not currently on treatment. The iRLS patients had greater accommodation in motor but not sensory axons to long-lasting hyperpolarization compared to age-matched healthy subjects, indicating greater inward rectification in iRLS. The most reasonable explanation is that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open at less hyperpolarized membrane potentials, a view supported by mathematical modelling. The half-activation potential for HCN channels (Bq) was the single best parameter that accounted for the difference between normal controls and iRLS data. A 6mV depolarization of Bq reduced the discrepancy between the normal control model and the iRLS data by 92.1%. Taken together, our results suggest an increase in the excitability of motor units in iRLS that could enhance the likelihood of leg movements. The abnormal axonal properties are consistent with other findings indicating that the peripheral system is part of the network involved in iRLS.

On the role of transient depolarization-activated K+ current in microvillar photoreceptors.

Photoreceptors in the compound eyes of most insect species express two functional types of depolarization-activated potassium currents: a transient A-type current (IA) and a sustained delayed rectifier current (IDR). The role of Shaker-dependent IA in Drosophila melanogaster photoreceptors was previously investigated by comparing intracellular recordings from Shaker and wild-type photoreceptors. Shaker channels were proposed to be involved in low-frequency signal amplification in dim light and reduction of the metabolic cost of information transfer. Here, I study the function of IA in photoreceptors of the cockroach Panchlora nivea using the patch-clamp method. Responses to Gaussian white-noise stimuli reveal that blockade of IA with 4-aminopyridine has no discernible effect on voltage responses or information processing. However, because open-channel blockers are often ineffective at low membrane potentials, no conclusion on the role of IA could be made on the basis of negative results of pharmacological tests. Using a relatively large set of control data, a physiological variability analysis was performed to discern the role of IA. Amplitudes of the IA window current and half-activation potentials correlate strongly with membrane corner frequencies, especially in dim light, indicating that IA facilitates transmission of higher frequencies. Consistent with voltage-dependent inactivation of IA, these correlations decrease with depolarization in brighter backgrounds. In contrast, correlations involving IDR are comparatively weak. Upon reexamining photoreceptor conductance in wild-type and Shaker strains of D. melanogaster, I find a biphasic voltage dependence near the resting potential in a minority of photoreceptors from both strains, indicating that Shaker channels are not crucial for early amplification of voltage signals in D. melanogaster photoreceptors. Leak current in Shaker photoreceptors at the level of the soma is not elevated. These results suggest a novel role for IA in facilitating transmission of high-frequency signals in microvillar photoreceptors.

Effects of isovitexin on transient outward potassium current of ventricular myocytes in rats

To investigate the effects of isovitexin Ⅳ on transient outward potassium current in rat ventricular myocytes. In this study, MTT assay was used to investigate the safe range of isovitexin. The results showed that the IC₅₀ of the drug was in the range of 10-30 μmol•L⁻¹, and the drug concentration of 1-3 μmol•L⁻¹ for the patch clamp test was within the safe range. In addition, the single ventricular myocytes were obtained by single-enzymatic hydrolysis through aortic retrograde perfusion. The transient outward potassium current (Ito) of rat ventricular myocytes was guided and measured by whole-cell patch-clamp technique and the changes of current characteristics were recorded after isovite was applied. When the concentration of IV was less than 0.1 μmol•L⁻¹, there was no significant effect on Ito. However, with the increase in the concentration of IV (≥0.3 μmol•L⁻¹), the peak of Ito was decreased gradually, from (32.32±2.9) pA/pF to （25.83±4.3） pA/pF, 1 μmol•L⁻¹ IV and (19.51±3.5) pA/pF, 3 μmol•L⁻¹ IV respectively, with an inhibition effect in a concentration-dependent manner. In the range of 1-3 μmol•L⁻¹, IV down-regulated the I-V curve of Ito significantly. The activation curve showed that IV can enable the maximum half activation potential (V1/2) to move to the positive direction, and the V1/2 was increased from (19.59±1.6) mV to (22.81±1.7) mV and (28.86±1.4) mV at concentration of 1, 3 μmol•L⁻¹, meanwhile the activation curve moved to the right. However, the maximum half inactivating potential (V1/2) of the steady-state inactivation curve of Ito was significantly decreased from (－51.43±0.99) mV to (－61.81±1.3) mV with concentration of 1 μmol•L⁻¹ and (－71.50±1.4) mV with concentration of 3 μmol•L⁻¹. The inactivation time constant of recovery from inactivation (τ) was up-regulated significantly from (94.89±0.73) ms to (118.5±1.5) ms and (162.4±1.4) ms at concentration of 1, 3 μmol•L⁻¹ respectively. Meanwhile IV could enable the inactivation recovery curve to move to the right, which suggested that it can prolong the recovery time from inactivation of the transient outward potassium channel. In conclusion, isovitexin had a high inhibitory effect on Ito in rat ventricular myocytes.

Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BKCa activator LDD175.

Plasma membrane hyperpolarization associated with activation of Ca2+-activated K+ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K+ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K+ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K+ current, and internal alkalinization and Ca2+ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K+ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca2+. In contrast, elevation of intracellular Ca2+ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca2+-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Acepromazine inhibits hERG potassium ion channels expressed in human embryonic kidney 293 cells.

The effects of acepromazine on human ether-à-go-go-related gene (hERG) potassium channels were investigated using whole-cell voltage-clamp technique in human embryonic kidney (HEK293) cells transfected with hERG. The hERG currents were recorded with or without acepromazine, and the steady-state and peak tail currents were analyzed for the evaluating the drug effects. Acepromazine inhibited the hERG currents in a concentration-dependent manner with an IC50 value of 1.5 µM and Hill coefficient of 1.1. Acepromazine blocked hERG currents in a voltage-dependent manner between –40 and +10 mV. Before and after application of acepromazine, the half activation potentials of hERG currents changed to hyperpolarizing direction. Acepromazine blocked both the steady-state hERG currents by depolarizing pulse and the peak tail currents by repolarizing pulse; however, the extent of blocking by acepromazine in the repolarizing pulse was more profound than that in the depolarizing pulse, indicating that acepromazine has a high affinity for the open state of the channels, with a relatively lower affinity for the closed state of hERG channels. A fast application of acepromazine during the tail currents inhibited the open state of hERG channels in a concentration-dependent. The steady-state inactivation of hERG currents shifted to the hyperpolarized direction by acepromazine. These results suggest that acepromazine inhibits the hERG channels probably by an open- and inactivated-channel blocking mechanism. Regarding to the fact that the hERG channels are the potential target of drug-induced long QT syndrome, our results suggest that acepromazine can possibly induce a cardiac arrhythmia through the inhibition of hERG channels.

Long-Term Potentiation at the Mossy Fiber-Granule Cell Relay Invokes Postsynaptic Second-Messenger Regulation of Kv4 Channels.

Cerebellar granule cells receive mossy fiber inputs that convey information on different sensory modalities and feedback from descending cortical projections. Recent work suggests that signal processing across multiple cerebellar lobules is controlled differentially by postsynaptic ionic mechanisms at the level of granule cells. We found that long-term potentiation (LTP) of mossy fiber input invoked a large increase in granule cell excitability by modifying the biophysical properties of Kv4 channels through a specific signaling cascade. LTP of granule cell output became evident in response to bursts of mossy fiber input, revealing that Kv4 control of intrinsic excitability is modified to respond most effectively to patterns of afferent input that are characteristic of physiological sensory patterns.

Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons

Lidocaine, which blocks voltage-gated sodium channels, is widely used in surgical anesthesia and pain management. Recently, it has been proposed that the hyperpolarization-activated cyclic nucleotide (HCN) channel is one of the other novel targets of lidocaine. Substantia gelatinosa in the spinal dorsal horn, which plays key roles in modulating nociceptive information from primary afferents, comprises heterogeneous interneurons that can be electrophysiologically categorized by firing pattern. Our previous study demonstrated that a substantial proportion of substantia gelatinosa neurons reveal the presence of HCN current (Ih); however, the roles of lidocaine and HCN channel expression in different types of substantia gelatinosa neurons remain unclear. By using the whole-cell patch-clamp technique, we investigated the effect of lidocaine on Ih in rat substantia gelatinosa neurons of acute dissociated spinal cord slices. We found that lidocaine rapidly decreased the peak Ih amplitude with an IC50 of 80 μM. The inhibition rate on Ih was not significantly different with a second application of lidocaine in the same neuron. Tetrodotoxin, a sodium channel blocker, did not affect lidocaine's effect on Ih. In addition, lidocaine shifted the half-activation potential of Ih from -109.7 to -114.9 mV and slowed activation. Moreover, the reversal potential of Ih was shifted by -7.5 mV by lidocaine. In the current clamp, lidocaine decreased the resting membrane potential, increased membrane resistance, delayed rebound depolarization latency, and reduced the rebound spike frequency. We further found that approximately 58% of substantia gelatinosa neurons examined expressed Ih, in which most of them were tonically firing. Our studies demonstrate that lidocaine strongly inhibits Ih in a reversible and concentration-dependent manner in substantia gelatinosa neurons, independent of tetrodotoxin-sensitive sodium channels. Thus, our study provides new insight into the mechanism underlying the central analgesic effect of the systemic administration of lidocaine.

Two heterozygous Cav3.2 channel mutations in a pediatric chronic pain patient: recording condition-dependent biophysical effects.

We report expression system-dependent effects of heterozygous mutations (P769L and A1059S) in the Cav3.2 CACNA1H gene identified in a pediatric patient with chronic pain and absence seizures. The mutations were introduced individually into recombinant channels and then analyzed by means of electrophysiology. When both mutants were co-expressed in tsA-201 cells, we observed a loss of channel function, with significantly smaller current densities across a wide range of voltages (-40 to +20 mV). In addition, when both mutant channels were co-expressed, the channels opened at a more depolarizing potential with a ~5-mV right shift in the half-activation potential, with no changes in half-inactivation potential and the rate of recovery from inactivation. Interestingly, when both mutants were co-expressed in the neuronal-derived CAD cells in a different extracellular milieu, the effect was remarkably different. Although not statistically significant (p < 0.07), current densities appeared augmented compared to wild-type channels and the difference in the half-activation potential was lost. This could be attributed to the replacement of extracellular sodium and potassium with tetraethylammonium chloride. Our results show that experimental conditions can be a confounding factor in the biophysical effects of T-type calcium channel mutations found in certain neurological disorders.

Effects of aging in Parkinson�s disease: Role of Ca channel in dopamine neuron computational model

Effects of aging in Parkinson�s disease: Role of Ca channel in dopamine neuron computational model

The Role of Met-691 in Heme-Dependent Regulation Supports the Presence of a Cytochrome-C-Like Structure in Human BK Channels

We have recently proposed that the linker region between RCK domains in the cytoplasmic portion of BK channels consists of a Cytochrome-c-like domain, which encompasses a conserved heme regulatory motif (HRM, 612CKACH616 (Tang et al., Nature 2003) and endows BK channels with catalytic properties (Yusifov et al., Biophys J 2710-Pos 2014). Alignment of the BK Cytochrome-c-like domain and the human Cytochrome C (CytC) sequences revealed that they share, in addition to the HRM, key structural and functional elements. Notably, Met-80 in CytC, which is the second axial ligand to the heme iron, aligns with BK Met-691. In fact, mutation M691A dramatically reduced the Heme-dependent peroxidase activity of the purified BK channel Gating Ring. We assessed the involvement M691 in the heme modulation of BK channel conducting properties by inside-out patch clamp in WT and M691A channels expressed in Xenopus oocytes. The K+ current from WT BK channels was reduced by 61.6±14.6% (140mV) in 100nM heme. On the contrary, under identical conditions, K+ current was reduced by only 13.2±2.23% in BK channels carrying the M691A mutation. Moreover, the conductance half-activation potential (Vhalf) of WT BK shifted from 81.2±3.03 to 126±1.67mV after the addition of 100nM heme. Conversely, in M691A channels, the Vhalf was largely unperturbed by 100nM heme (from 98.2±4.13 to 100±8.76mV). Thus, M691A essentially eliminated the BK channel sensitivity to heme. These results reinforce the view that a Cytochrome-c-like domain, which associates heme via the HRM and a second axial ligand (Met-691) is an integral part of BK channels.

Effects of aging in Parkinson�s disease: Role of L - type Ca channel in dopamine neuron computational model

Effects of aging in Parkinson�s disease: Role of L - type Ca channel in dopamine neuron computational model

Functional contributions of HCN channels in the primary auditory neurons of the mouse inner ear

The hyperpolarization-activated current, Ih, is carried by members of the Hcn channel family and contributes to resting potential and firing properties in excitable cells of various systems, including the auditory system. Ih has been identified in spiral ganglion neurons (SGNs); however, its molecular correlates and their functional contributions have not been well characterized. To investigate the molecular composition of the channels that carry Ih in SGNs, we examined Hcn mRNA harvested from spiral ganglia of neonatal and adult mice using quantitative RT-PCR. The data indicate expression of Hcn1, Hcn2, and Hcn4 subunits in SGNs, with Hcn1 being the most highly expressed at both stages. To investigate the functional contributions of HCN subunits, we used the whole-cell, tight-seal technique to record from wild-type SGNs and those deficient in Hcn1, Hcn2, or both. We found that HCN1 is the most prominent subunit contributing to Ih in SGNs. Deletion of Hcn1 resulted in reduced conductance (Gh), slower activation kinetics (τfast), and hyperpolarized half-activation (V1/2) potentials. We demonstrate that Ih contributes to SGN function with depolarized resting potentials, depolarized sag and rebound potentials, accelerated rebound spikes after hyperpolarization, and minimized jitter in spike latency for small depolarizing stimuli. Auditory brainstem responses of Hcn1-deficient mice showed longer latencies, suggesting that HCN1-mediated Ih is critical for synchronized spike timing in SGNs. Together, our data indicate that Ih contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.

Molecular and cellular influences of permethrin on mammalian nociceptors at physiological temperatures

The influence of pyrethroid insecticides is thought to be abrogated at mammalian physiological temperatures. Yet there are many reports of transient pain and paresthesia following accidental exposures. Using whole cell patch clamp techniques, we examined the interaction of the pyrethroid insecticide permethrin on skin, muscle and putative vascular nociceptors of the rat DRG (dorsal root ganglion). Following permethrin (10μM) application, action potential (AP) duration was increased in all nociceptor populations, but only muscle nociceptors developed spontaneous activity or increased excitability (tests at 21°C). TTX (tetrodotoxin) did not prevent the development of spontaneous activity or reduce excitability. We examined the influence of permethrin on TTX resistant channel proteins that control excitability and spontaneous activity (Nav1.8, voltage-gated sodium channel 1.8; Kv7, voltage gated potassium channel 7). In all nociceptor populations, permethrin increased the tau of deactivation (taudeact), in a voltage dependent manner, and hyperpolarized the V1/2 for activation over 10mV. There were no permethrin dependent influences on Kv7, or on the voltage dependence of inactivation of Nav1.8. The influence of permethrin on AP duration, after hyperpolarization, spontaneous activity, half-activation potential (V1/2) and taudeact were reduced, but not fully reversed, when tests were conducted at 35°C. In conclusion, permethrin greatly modifies the voltage dependent activation and deactivation of Nav1.8 expressed in skin, muscle and vascular nociceptors. These influences remain significant at 35°C. One population of muscle nociceptors exhibited a unique vulnerability to the acute administration of permethrin manifested as increased excitability and spontaneous activity.