Effect of ABT-639 on Cav3.2 channel activity and its analgesic actions in mouse models of inflammatory and neuropathic pain.

Cav3.2 T-type calcium channels are important targets for pain relief in rodent models of inflammatory and neuropathic pain. Even though many T-type channel blockers have been tested in mice, only one molecule, ABT-639, has been tested in phase II clinical studies and did not produce analgesic effects over placebo. Here we examined the effects of ABT-639 on Cav3.2 channel activity in tsA-201 cells and dorsal root ganglion (DRG) neurons, in comparison with another established Cav3.2 inhibitor Z944. These experiments revealed that Z944 mediated ∼100-fold more potent inhibition of Cav3.2 currents than ABT-639, with the latter blocking channel activity by less than 15 percent when applied at a concentration of 30 µM. A slight increase in ABT-639 potency was observed at more depolarized holding potentials, suggesting that this compound may act preferentially on inactivated channels. We tested the effects of both compounds in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain, and in partial sciatic nerve injury model of neuropathic pain in mice. In the neuropathic pain model, both Z944 and ABT-639 reversed mechanical hypersensitivity to similar degrees when delivered systemically, but remarkably, when delivered intrathecally, only Z944 was effective. In the CFA model, both compounds reversed thermal hyperalgesia upon systemic delivery, but only Z944 mediated pain relief upon intrathecal delivery, indicating that ABT-639 acts primarily at peripheral sites. ABT-639 lost its analgesic effects in CFA treated Cav3.2 null mice, indicating that these channels are essential for ABT-639-mediated pain relief despite its poor inhibition of Cav3.2 currents.

G-protein tonic inhibition of calcium channels in pancreatic ß-cells.

Voltage-gated calcium channels (CaV) conduct Ca2+ influx promoting neurotransmitters and hormone release. CaV are finely regulated by voltage-dependent and independent pathways either by G-protein-coupled receptors (GPCRs) or intramembrane lipids, respectively, in neurons and glands. Interestingly, pancreatic ß-cells are abundantly innervated by both sympathetic and parasympathetic neurons, while a variety of high-voltage activated (HVA) Ca2+ channels are present in these cells. Thus, autonomic system seems to exert a tonic inhibition on HVA Ca2+ channels throughout GPCRs, constitutively preventing hormone secretion. Therefore, this work aimed to investigate noradrenergic and cholinergic inhibition of HVA Ca2+ channels in pancreatic ß-cells. Experiments were conducted in pancreatic ß-cells of rat by using patch-clamping methods, immunocytochemistry, pharmacological probes, and biochemical reagents. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask tonic inhibition. Herein, we consistently find a basal tonic inhibition of HVA Ca2+ channels according to a GPCRs regulation. Facilitation ratio is enhanced by noradrenaline (NA) according to a voltage-dependent regulation and a membrane-delimited mechanism, while no facilitation changes are observed with carbachol or phosphatidylinositol 4,5-bisphosphate (PIP2). Furthermore, carbachol or intramembrane lipids, such as PIP2, do not change facilitation ratio according to a voltage-independent regulation. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs, suggesting a natural brake preventing cells from exhaustive insulin secretion.NEW & NOTEWORTHY Our results support the hypothesis that GPCRs tonically inhibit HVA Ca2+ channels in pancreatic ß-cells. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask voltage-dependent inhibition of Ca2+ channels. The novelty of these results strengthens the critical role of Gßγ's in Ca2+ channel regulation, highlighting kinetic slowing and increased facilitation ratio. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs underlying fine-tuning modulation of insulin secretion.

Inhibition of high-voltage-activated calcium currents by acute hypoxia in cultured retinal ganglion cells.

Hypoxia is a common factor of numerous ocular diseases that lead to dysfunctions and loss of retinal ganglion cells (RGCs) with subsequent vision loss. High-voltage-activated calcium channels are the main source of calcium entry into neurons. Their activity plays a central role in different signaling processes in health and diseases, such as enzyme activation, gene transcription, synaptic transmission, or the onset of cell death. This study aims to establish and evaluate the initial effect of the early stage of acute hypoxia on somatic HVA calcium currents in cultured RGCs. HVA calcium currents were recorded in RGCs using the whole-cell patch-clamp technique in the voltage-clamp mode. The fast local superfusion was used for a brief (up to 270 s) application of the hypoxic solution (pO2 < 5 mmHg). The switch from normoxic to hypoxic solutions and vice versa was less than 1 s. The HVA calcium channel activity was inhibited by acute hypoxia in 79% of RGCs (30 of 38 RGCs) in a strong voltage-dependent manner. The level of inhibition was independent of the duration of hypoxia or repeated applications. The hypoxia-induced inhibition of calcium currents had a strong correlation with the duration of hypoxia and showed the transition from reversible to irreversible at 75 s of hypoxia and longer. The results obtained are the first demonstration of the phenomena of HVA calcium current inhibition by acute hypoxia in RGCs and provide a conceptual framework for further research.

O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents.

Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 µM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

Fat-Diets in Perinatal Stages Altered Nr3c2-Mediated Ca2+ Currents in Mesenteric Arteries of Offspring Rats.

SCOPE: Perinatal high-fat diets (PHF) can influence fetal/neonate development, resulting in cardiovascular pathogenesis, but precise mechanisms remain unclear. This study tests aldosterone receptor-mediated Ca2+ influx and the underlying mechanisms influenced by PHF. METHODS AND RESULTS: Maternal S.D. rats receive PHF during pregnancy and lactation periods. Their male offspring are fed normal diets after weaning for four months. Mesenteric arteries (MA) are for electrophysiological testing, Ca2+ imaging, target gene expression, and promotor methylation. PHF increases aldosterone receptor gene Nr3c2-mediated Ca2+ currents in the smooth muscle cells (SMCs) of the MA via L-type Ca2+ channels (LTCC) in the offspring. The increased expression of aldosterone-receptors and LTCC are responsible for an activated Nr3c2-LTCC pathway in the vasculature, eventually predisposes an increase of Ca2+ influx in the myocytes of resistance arteries. The inhibitor of aldosterone-receptors suppresses the increased Ca2+ currents in the SMCs. Nr3c2 and LTCC are upregulated through the transcriptional mechanism in methylation, which can be reversed in the functional changes by methylation inhibitor 5AZA. CONCLUSION: The results firstly demonstrate that aldosterone-receptor activation can stimulate Ca2+ currents via LTCC in vascular myocytes, which can be altered by perinatal foods via epigenetic changes of DNA methylation in the promoters of Nr3c2 and LTCC.

Aluminum alters excitability by inhibiting calcium, sodium, and potassium currents in bovine chromaffin cells.

Aluminum (Al3+ ) has long been related to neurotoxicity and neurological diseases. This study aims to describe the specific actions of this metal on cellular excitability and neurotransmitter release in primary culture of bovine chromaffin cells. Using voltage-clamp and current-clamp recordings with the whole-cell configuration of the patch clamp technique, online measurement of catecholamine release, and measurements of [Ca2+ ]c with Fluo-4-AM, we have observed that Al3+ reduced intracellular calcium concentrations around 25% and decreased catecholamine secretion in a dose-dependent manner, with an IC50 of 89.1 µM. Al3+ blocked calcium currents in a time- and concentration-dependent manner with an IC50 of 560 µM. This blockade was irreversible since it did not recover after washout. Moreover, Al3+ produced a bigger blockade on N-, P-, and Q-type calcium channels subtypes (69.5%) than on L-type channels subtypes (50.5%). Sodium currents were also inhibited by Al3+ in a time- and concentration-dependent manner, 24.3% blockade at the closest concentration to the IC50 (399 µM). This inhibition was reversible. Voltage-dependent potassium currents were low affected by Al3+ . Nonetheless, calcium/voltage-dependent potassium currents were inhibited in a concentration-dependent manner, with an IC50 of 447 µM. This inhibition was related to the depression of calcium influx through voltage-dependent calcium channels subtypes coupled to BK channels. In summary, the blockade of these ionic conductance altered cellular excitability that reduced the action potentials firing and so, the neurotransmitter release and the synaptic transmission. These findings prove that aluminum has neurotoxic properties because it alters neuronal excitability by inhibiting the sodium currents responsible for the generation and propagation of impulse nerve, the potassium current responsible for the termination of action potentials, and the calcium current responsible for the neurotransmitters release.

Beta-blocker treatment of patients with atrial fibrillation attenuates spontaneous calcium release-induced electrical activity.

AIMS: Atrial fibrillation (AF) has been associated with excessive spontaneous calcium release, linked to cyclic AMP (cAMP)-dependent phosphorylation of calcium regulatory proteins. Because ß-blockers are expected to attenuate cAMP-dependent signaling, we aimed to examine whether the treatment of patients with ß-blockers affected the incidence of spontaneous calcium release events or transient inward currents (ITI). METHODS: The impact of treatment with commonly used ß-blockers was analyzed in human atrial myocytes from 371 patients using patch-clamp technique, confocal calcium imaging or immunofluorescent labeling. Data were analyzed using multivariate regression analysis taking into account potentially confounding effects of relevant clinical factors RESULTS: The L-type calcium current (ICa) density was diminished significantly in patients with chronic but not paroxysmal AF and the treatment of patients with ß-blockers did not affect ICa density in any group. By contrast, the ITI frequency was elevated in patients with either paroxysmal or chronic AF that did not receive treatment, and ß-blocker treatment reduced the frequency to levels observed in patients without AF. Confocal calcium imaging showed that ß-blocker treatment also reduced the calcium spark frequency in patients with AF to levels observed in those without AF. Furthermore, phosphorylation of the ryanodine receptor (RyR2) at Ser-2808 and phospholamban at Ser-16 was significantly lower in patients with AF that received ß-blockers. CONCLUSION: Together, our findings demonstrate that ß-blocker treatment may be of therapeutic utility to prevent spontaneous calcium release-induced atrial electrical activity; especially in patients with a history of paroxysmal AF displaying preserved ICa density.

Similar Presynaptic Action Potential-Calcium Influx Coupling in Two Types of Large Mossy Fiber Terminals Innervating CA3 Pyramidal Cells and Hilar Mossy Cells.

Morphologically similar axon boutons form synaptic contacts with diverse types of postsynaptic cells. However, it is less known to what extent the local axonal excitability, presynaptic action potentials (APs), and AP-evoked calcium influx contribute to the functional diversity of synapses and neuronal activity. This is particularly interesting in synapses that contact cell types that show only subtle cellular differences but fulfill completely different physiological functions. Here, we tested these questions in two synapses that are formed by rat hippocampal granule cells (GCs) onto hilar mossy cells (MCs) and CA3 pyramidal cells, which albeit share several morphologic and synaptic properties but contribute to distinct physiological functions. We were interested in the deterministic steps of the action potential-calcium ion influx coupling as these complex modules may underlie the functional segregation between and within the two cell types. Our systematic comparison using direct axonal recordings showed that AP shapes, Ca2+ currents and their plasticity are indistinguishable in synapses onto these two cell types. These suggest that the complete module that couples granule cell activity to synaptic release is shared by hilar mossy cells and CA3 pyramidal cells. Thus, our findings present an outstanding example for the modular composition of distinct cell types, by which cells employ different components only for those functions that are deterministic for their specialized functions, while many of their main properties are shared.

ADAM17 Mediates Proteolytic Maturation of Voltage-Gated Calcium Channel Auxiliary α2δ Subunits, and Enables Calcium Current Enhancement.

The auxiliary α2δ subunits of voltage-gated calcium (CaV) channels are key to augmenting expression and function of CaV1 and CaV2 channels, and are also important drug targets in several therapeutic areas, including neuropathic pain. The α2δ proteins are translated as preproteins encoding both α2 and δ, and post-translationally proteolyzed into α2 and δ subunits, which remain associated as a complex. In this study, we have identified ADAM17 as a key protease involved in proteolytic processing of pro-α2δ-1 and α2δ-3 subunits. We provide three lines of evidence: First, proteolytic cleavage is inhibited by chemical inhibitors of particular metalloproteases, including ADAM17. Second, proteolytic cleavage of both α2δ-1 and α2δ-3 is markedly reduced in cell lines by knockout of ADAM17 but not ADAM10. Third, proteolytic cleavage is reduced by the N-terminal active domain of TIMP-3 (N-TIMP-3), which selectively inhibits ADAM17. We have found previously that proteolytic cleavage into mature α2δ is essential for the enhancement of CaV function, and in agreement, knockout of ADAM17 inhibited the ability of α2δ-1 to enhance both CaV2.2 and CaV1.2 calcium currents. Finally, our data also indicate that the main site of proteolytic cleavage of α2δ-1 is the Golgi apparatus, although cleavage may also occur at the plasma membrane. Thus, our study identifies ADAM17 as a key protease required for proteolytic maturation of α2δ-1 and α2δ-3, and thus a potential drug target in neuropathic pain.

Potential Benefits of Flavonoids on the Progression of Atherosclerosis by Their Effect on Vascular Smooth Muscle Excitability.

Flavonoids are a group of secondary metabolites derived from plant-based foods, and they offer many health benefits in different stages of several diseases. This review will focus on their effects on ion channels expressed in vascular smooth muscle during atherosclerosis. Since ion channels can be regulated by redox potential, it is expected that during the onset of oxidative stress-related diseases, ion channels present changes in their conductive activity, impacting the progression of the disease. A typical oxidative stress-related condition is atherosclerosis, which involves the dysfunction of vascular smooth muscle. We aim to present the state of the art on how redox potential affects vascular smooth muscle ion channel function and summarize if the benefits observed in this disease by using flavonoids involve restoring the ion channel activity.

A novel proline-rich M-superfamily conotoxin that can simultaneously affect sodium, potassium and calcium currents.

BACKGROUND: Conotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. METHODS: Vr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells. RESULTS: About 10 µM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 µM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 µM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 µM. Moreover, 10 µM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents. CONCLUSIONS: Vr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.

Detection of Root Physiological Parameters and Potassium and Calcium Currents in the Rhizoplane of the Apple Rootstock Superior Line 12-2 With Improved Apple Replant Disease Resistance.

The cultivation of resistant rootstocks is one of the more effective ways to mitigate apple replant disease (ARD). We performed an ion current test, a pot experiment, and a pathogen infection test on the apple rootstocks 12-2 (self-named), T337, and M26. The ion current test showed that exposure to ARD soil extract for 30 min had a significant effect on K+ ion currents at the meristem, elongation, and mature zones of the M26 rhizoplane and on Ca2+ currents in the meristem and elongation zones. ARD also had a significant effect on Ca2+ currents in the meristem, elongation, and mature zones of the T337 rhizoplane. Exposure to ARD soil extract for 5 min had a significant effect on K+ currents in the meristem, elongation, and mature zones of 12-2 and on the Ca2+ currents in the elongation and mature zones. Compared to a 5-min exposure, a 30-min exposure to ARD extract had a less pronounced effect on K+ and Ca2+ currents in the 12-2 rhizoplane. The pot experiment showed that ARD soil had no significant effect on any root architectural or physiological parameters of 12-2. By contrast, ARD soil significantly reduced some root growth indices and the dry and fresh weights of T337 and M26 compared with controls on sterilized soil. ARD also had a significant effect on root metabolic activity, root antioxidant enzyme activity (except superoxide dismutase for T337), and malondialdehyde content of T337 and M26. Pathogen infection tests showed that Fusarium proliferatum MR5 significantly affected the root structure and reduced the root metabolic activity of T337 and M26. It also reduced their root antioxidant enzyme activities (except catalase for T337) and significantly increased the root malondialdehyde content, reactive oxygen levels, and proline and soluble sugar contents. By contrast, MR5 had no such effects on 12-2. Based on these results, 12-2 has the potential to serve as an important ARD-resistant rootstock.

A novel proline-rich M-superfamily conotoxin that can simultaneously affect sodium, potassium and calcium currents

Background Conotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. Methods Vr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells. Results About 10 μM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 μM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 μM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 μM. Moreover, 10 μM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents. Conclusions Vr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.(AU)

A GLP-1/GIP/Gcg receptor triagonist improves memory behavior, as well as synaptic transmission, neuronal excitability and Ca2+ homeostasis in 3xTg-AD mice.

Alzheimer's disease (AD) is a progressively neurodegenerative disorder, which seriously affects human health and cannot be stopped by current treatments. Type 2 diabetes mellitus (T2DM) is a risk factor for AD. Our recent studies reported the neuroprotective effects of a GLP-1/GIP/Glucagon receptor triagonist (Triagonist), a novel unimolecular anti-diabetic drug, in cognitive and pathological improvements of 3xTg-AD mice. However, the detailed electrophysiological and molecular mechanisms underlying neuroprotection remain unexplored. The present study investigated the underlying electrophysiological and molecular mechanisms further by using whole-cell patch clamp techniques. Our results revealed that chronic Triagonist treatment effectively reduced working memory and reference memory errors of 3xTg-AD mice in a radial maze test. In addition, the Triagonist increased spontaneous excitatory synaptic activities, differentially modulated voltage- and chemically-gated Ca2+ flux, and reduced the over-excitation of pyramidal neurons in hippocampal slices of 3xTg-AD mice. In addition, chronic Triagonist treatment also up-regulated the expression levels of synaptophysin and PSD-95 in the hippocampus of 3xTg-AD mice. These results indicate that the Triagonist could improve memory formation, as well as synaptic transmission, Ca2+ balance, and neuronal excitability in 3xTg-AD mice. These neuroprotective effects of Triagonist may be involved in the up-regulation of synaptophysin and PSD-95. Therefore, the study suggests that multi-receptor agonists might be a novel therapeutic strategy for the treatment of AD.

Differential effects of HDAC inhibitors on PPN oscillatory activity in vivo.

The pedunculopontine nucleus (PPN) has long been known to be part of the reticular activating system (RAS) in charge of arousal and REM sleep. We previously showed that in vitro exposure to a HDAC Class I and II mixed inhibitor (TSA), or a specific HDAC class IIa inhibitor (MC 1568), decreased PPN gamma oscillations. Given the lack of information on systemic in vivo treatments on neuronal synaptic properties, the present study was designed to investigate the systemic effect of HDAC inhibitors (HDACi) on PPN rhythmicity. Rat pups were injected (acute, single dose) with TSA (4 or 20 mg/kg), MC1568 (4 or 20 mg/kg), or MS275 (20 or 100 mg/kg). Our results show that MC1568 (20 mg/kg) reduced mean frequency of PPN oscillations at gamma band, while increasing mean input resistance (Rm) of PPN neurons. For TSA (4 and 20 mg/kg), we observed reduced mean frequency of oscillations at gamma band and increased mean Rm of PPN neurons. Systemic administration of 20 mg/kg MC1568 and TSA effects on Rm were washed out after 60 min of in vitro incubation of PPN slices, suggesting an underlying functional recovery of PPN calcium-mediated gamma band oscillations over time. In addition, at a lower dose, 4 mg/kg, MC1568 and TSA induced higher mean amplitude gamma oscillations. Blocking HDAC class I might not have deleterious effects on gamma activity, but, more importantly, the inhibition of HDAC class I (at 100 mg/kg) increased gamma band oscillations. In conclusion, the present results in vivo validate our previous findings in vitro and further expand information on the effects of HDAC inhibition on PPN rhythmicity. PPN neurons require normal activity of HDAC class IIa in order to oscillate at gamma band.

Gamma oscillations in the pedunculopontine nucleus are regulated by F-actin: neuroepigenetic implications.

The pedunculopontine nucleus (PPN) is part of the reticular activating system (RAS) in charge of arousal and rapid eye movement sleep. The presence of high-frequency membrane oscillations in the gamma-band range in the PPN has been extensively demonstrated both in vivo and in vitro. Our group previously described histone deacetylation (HDAC) inhibition in vitro induced protein changes in F-actin cytoskeleton and intracellular Ca2+ concentration regulation proteins in the PPN. Here, we present evidence that supports the presence of a fine balance between HDAC function and calcium calmodulin kinase II-F-actin interactions in the PPN. We modified F-actin polymerization in vitro by using jasplakinolide (1 µM, a promoter of F-actin stabilization), or latrunculin-B (1 µM, an inhibitor of actin polymerization). Our results showed that shifting the balance in either direction significantly reduced PPN gamma oscillation as well as voltage-dependent calcium currents.

Imaging Native Calcium Currents in Brain Slices.

Imaging techniques may overcome the limitations of electrode techniques to measure locally not only membrane potential changes, but also ionic currents. Here, we review a recently developed approach to image native neuronal Ca2+ currents from brain slices. The technique is based on combined fluorescence recordings using low-affinity Ca2+ indicators possibly in combination with voltage sensitive dyes. We illustrate how the kinetics of a Ca2+ current can be estimated from the Ca2+ fluorescence change and locally correlated with the change of membrane potential, calibrated on an absolute scale, from the voltage fluorescence change. We show some representative measurements from the dendrites of CA1 hippocampal pyramidal neurons, from olfactory bulb mitral cells and from cerebellar Purkinje neurons. We discuss the striking difference in data analysis and interpretation between Ca2+ current measurements obtained using classical electrode techniques and the physiological currents obtained using this novel approach. Finally, we show how important is the kinetic information on the native Ca2+ current to explore the potential molecular targets of the Ca2+ flux from each individual Ca2+ channel.

Disruption of the Key Ca2+ Binding Site in the Selectivity Filter of Neuronal Voltage-Gated Calcium Channels Inhibits Channel Trafficking.

Voltage-gated calcium channels are exquisitely Ca2+ selective, conferred primarily by four conserved pore-loop glutamate residues contributing to the selectivity filter. There has been little previous work directly measuring whether the trafficking of calcium channels requires their ability to bind Ca2+ in the selectivity filter or to conduct Ca2+. Here, we examine trafficking of neuronal CaV2.1 and 2.2 channels with mutations in their selectivity filter and find reduced trafficking to the cell surface in cell lines. Furthermore, in hippocampal neurons, there is reduced trafficking to the somatic plasma membrane, into neurites, and to presynaptic terminals. However, the CaV2.2 selectivity filter mutants are still influenced by auxiliary α2δ subunits and, albeit to a reduced extent, by ß subunits, indicating the channels are not grossly misfolded. Our results indicate that Ca2+ binding in the pore of CaV2 channels may promote their correct trafficking, in combination with auxiliary subunits. Furthermore, physiological studies utilizing selectivity filter mutant CaV channels should be interpreted with caution.

Effect of heat stress and Hsp90 inhibition on T-type calcium currents and voltage-dependent potassium currents in leydig cells.

Heat can trigger testicular damage and impair fertility. Leydig cells produce testosterone in response to stimulation by luteinizing hormone (LH), which induces Ca2+ entry and K+ efflux through ion channels in their plasma membrane. Considering that mechanisms coordinating the Leydig cell responses to hyperthermic stress remain unclear; the present study analyzed the effects of heat stress (HS, 43°C, 15 min) and inhibition of Hsp90 on T-type calcium currents and voltage-dependent potassium currents (VKC) in mice Leydig cells. Results show that HS reduced the VKC steady state currents at +80 mV (45.3%) and maximum conductance (71.5%), as well as increased the activation time constant (31.7%) and the voltage for which half the channels are open (30%). Hsp90 inhibition did not change the VKC currents. T-type calcium currents were not affected by HS or Hsp90 inhibition. In conclusion, HS can slow the activation, reduce the currents and voltage dependence of the VKC, suggesting a possible role of these currents in the response to hyperthermic stress in Leydig cells.

Synergism of myocardial ß-adrenoceptor blockade and I1-imidazoline receptor-driven signaling: Kinase-phosphatase switching.

Recently identified imidazoline receptors of the first type (I1Rs) on the cardiomyocyte's sarcolemma open a new field in calcium signaling research. In particular, it is interesting to investigate their functional interaction with other well-known systems, such as ß-adrenergic receptors. Here we investigated the effects of I1Rs activation on L-type voltage-gated Ca2+-currents under catecholaminergic stress induced by the application of ß-agonist, isoproterenol. Pharmacological agonist of I1Rs (I1-agonist), rilmenidine, and the putative endogenous I1-ligand, agmatine, have been shown to effectively reduce Ca2+-currents potentiated by isoproterenol. Inhibitory analysis shows that the ability to suppress voltage-gated Ca2+-currents by rilmenidine and agmatine is fully preserved in the presence of the protein kinase A blocker (PKA), which indicates a PKA-independent mechanism of their action. The blockade of NO synthase isoforms with 7NI does not affect the intrinsic effects of agmatine and rilmenidine, which suggests NO-independent signaling pathways triggered by I1Rs. A nonspecific serine/threonine protein phosphatase (STPP) inhibitor, calyculin A, abrogates effects of rilmenidine or agmatine on the isoproterenol-induced Ca2+-currents. Direct measurements of phosphatase activity in the myocardial tissues showed that activation of the I1Rs leads to stimulation of STPP, which could be responsible for the I1-agonist influences. Obtained data clarify peripheral effects that occur during activation of the I1Rs under endogenous catecholaminergic stress, and can be used in clinical practice for more precise control of heart contractility in some cardiovascular pathologies.