An alpha 40 subunit of a GTP-binding protein immunologically related to Go mediates a dopamine-induced decrease of Ca2+ current in snail neurons

Dopamine induces a decrease in voltage-dependent Ca2+ current in identified neurons of the snail H. aspersa. This effect is blocked by intracellular injection of activated B. pertussis toxin and of an affinity-purified antibody against the alpha subunit of bovine Go protein. The dopamine effect is mimicked by intracellular injection of mammalian alpha o. In snail nervous tissue, pertussis toxin ADP-ribosylates a single protein band on SDS gels, and this band is recognized in immunoblots by the anti-alpha o antibody. We propose that this is a 40 kd alpha subunit of a molluscan G protein immunologically related to alpha o and that it mediates the effect of dopamine on Ca2+ currents in identified snail neurons.     

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

Background

Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na⁺ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.

Results

Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na⁺ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Na_v1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Na_v1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.

Conclusion

Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Na_v1.3 subunit, and the function of the Na_v1.3 channel is regulated by PKC.     

Activation process of calcium-dependent potassium channel in Euhadra neurons: involvement of calcium/calmodulin and subsequent protein phosphorylation.

1. The activation process of Ca(2+)-dependent potassium channel was studied electrophysiologically and pharmacologically using identified neurons of the land snail, Euhadra peliomphala. 2. Ca(2+)-mediated delayed outward K current (IKD) was dose-dependently reduced by the calmodulin inhibitors, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, week) and N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide (W-7, potent). These antagonists also caused a slight membrane depolarization and increase in impulse discharge frequency with decrease in the amplitude of both action potential and after hyperpolarization. 3. The cAMP-dependent protein kinase inhibitor N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide (H-8) did not produce any significant effect on IKD and membrane potential. 4. Calmodulin, when injected into the neuron which had been treated with either W-5 or W-7, transiently restored the suppressed IKD nearly to the pretreatment level, and caused hyperpolarization of the cell. In contrast, calcium chloride, intracellularly injected in the same way, had little effect on both the IKD and the membrane potential shifted by these antagonists. 5. Intracellular injection of kinase II, a Ca2+/calmodulin-dependent protein kinase, caused an increase in the IKD and membrane hyperpolarization. Similar but weak effects were produced when a catalytic subunit (CS) of cAMP-dependent protein kinase was intracellularly injected. However, the neurons pretreated with W-7 no longer had any detectable increase in the IKD and hyperpolarization of the membrane. 6. These results suggest the possibility that Ca2+/camodulin-dependent protein phosphorylation may finally mediate the activation of a certain number of potassium channels.     

Calcium currents recorded from a neuronal α1C L-type calcium channel in Xenopus oocytes

Xenopus oocytes expressing neuronal α_1C, α₂ and β_1b calcium channel subunit cDNAs were used in this study. During two-electric voltage clamp recording the oocyte was injected with 10–20 nl of a 100 mM BAPTA solution. Under these conditions, the endogenous Ca-activated Cl current was completely suppressed resulting in an α_1C Ba current free from Cl current contamination. BAPTA injection also allowed α_1C currents with different permeating ions, including Ca, to be examined. Compared to Ba and Sr, α_1C whole cell Ca currents were smaller in magnitude and showed kinetic and voltage-dependent properties more similar to those for L-type Ca currents recorded in native cells. That Ca-dependent inactivation occurs in BAPTA-buffered cells suggests that the Ca-binding site involved in this type of inactivation is very close to the pore of the channel.     

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors

GABA_A receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α₅ has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α₅ subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α₅ subunit-containing GABA_A receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α_4/6 subunit-containing GABA_A receptors, but is sensitive to L-655708, an antagonist of the GABA_A receptors containing α₅ subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α₅ subunit in the turtle spinal cord. Together, these results suggest that GABA_A receptors containing the α₅ subunit mediate the tonic inhibitory currents observed in VHIs.     

Regulation of potential-dependent L-type Ca2+ currents by agmatine. Imidazoline receptors in isolated cardiomyocytes

The main goal of the present work was to study the mechanisms of voltage-gated L-type Ca²⁺ currents regulation by agmatine in isolated cardiomyocytes and to determine whether agmatine is involved in mediating the “arginine paradox”. It was shown that agmatine at concentrations from 200 μM to 15 mM inhibited L-type Ca²⁺ currents in isolated cardiomyocytes in a dose-dependent manner. The selective antagonists of α₂-adrenoceptors (α₂-ARs), yohimbine and rauwolscine, did not modulate the effect of agmatine. In contrast, efaroxan and idazoxan known to antagonize both α₂-ARs and type 1 imidazoline receptors (I₁Rs) decreased the efficiency of agmatine almost twofold. The NO synthase inhibitor 7NI insignificantly influenced the suppressive action of agmatine on L-type Ca²⁺ currents, whereas the protein kinase C inhibitor, calphostin C, markedly reduced the effects of agmatine. Arginine did not affect L-type Ca²⁺ currents in the presence of agmatine and vice versa. These data suggest that agmatine is not involved in mediating the “arginine paradox” and that its effects are not due to the activation of endothelial NO synthase (eNOS) followed by cGMP-dependent inhibition of L-type Ca²⁺ current. Most likely, agmatine acts via I₁Rs coupled with the signaling pathway that involves the activation of protein kinase C. Previously nothing was known about possible localization of I₁Rs in isolated cardiomyocytes. Consistently, we have shown that single cardiomyocytes express the nischarin genes homologous to the IRAS gene, which is considered in the modern literature as the major candidate for the gene encoding I₁Rs. To the best our knowledge, this is the first demonstration of I₁Rs expression at the level of individual cells, including cardiomyocytes.     

Study of effects of antibody to protein S100 on ionic channels of input and output currents of identified neurons of the snail Helix lucorum

In the course of electrophysiological experiments, two types of the neurons of the edible snail Helix lucorum were detected, which responded by different way to application of antibodies to the neuron-specific calcium-binding S 100 protein (AS1000). Under effect of AS100, frequency of the action potential (AP) generation in the spontaneously active V1, V3, V17, and RPa6 cells decreased, whereas in V4 and V6 cells increased. On addition of quinine solution the AP generation frequency of these neurons decreased more than twice, while the AP duration (t _S) rose 6 times. The combined action of AS100 and quinine did not change statistically significantly the AP generation frequency, membrane potential (MP) and AP generation threshold (AP_t), as compared with the effect of AS100 in saline. The value of the AP duration (t _S) increased 1.6 times, which was less pronounced as compared with the quinine action in saline. This means that AS100 prevents an increase of the AP duration after the quinine application (block of the Ca-depended K-channels). The main AS100 effect at the level of the ionic currents is shown to consist in a decrease of the maximal value of the input current, on average, by 20%, while of the output current, on average, by 12%.     

Upregulation of casein kinase 1ε in dorsal root ganglia and spinal cord after mouse spinal nerve injury contributes to neuropathic pain

Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-β-estradiol (E₂) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (I _Gly). In outside-out membrane patches from HIP neurons devoid of nuclei, E₂ similarly inhibited I _Gly, suggesting a non-genomic characteristic. Moreover, the E₂ effect on I _Gly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERα and ERβ) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E₂ on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E₂ only affected the I _Gly in cells expressing α2, α2β or α3β subunits, suggesting that either α2-containing or α3β-GlyRs mediate the E₂ effect observed in neurons. Furthermore, E₂ inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR α2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E₂ which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.     

α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Oxaliplatin Modulates the Characteristics of Voltage-Gated Calcium Channels and Action Potentials in Small Dorsal Root Ganglion Neurons of Rats

Oxaliplatin is important for treating colorectal cancer. Although oxaliplatin is highly effective, it has severe side effects, of which neurotoxicity in dorsal root ganglion (DRG) neurons is one of the most common. The key mechanisms of this neurotoxicity are still controversial. However, disturbances of calcium homeostasis in DRG neurons have been suggested to mediate oxaliplatin neurotoxicity. By using whole-cell patch-clamp and current-clamp techniques, as well as immunocytochemical staining, we examined the influence of short- and long-term exposure to oxaliplatin on voltage-gated calcium channels (VGCC) and different VGCC subtypes in small DRG neurons of rats in vitro. Exposure to oxaliplatin reduced VGCC currents (I_Ca(V)) in a concentration-dependent manner (1–500 μM; 13.8–63.3%). Subtype-specific measurements of VGCCs showed differential effects on I_Ca(V). While acute treatment with oxaliplatin led to a reduction in I_Ca(V) for P/Q-, T-, and L-type VGCCs, I_Ca(V) of N-type VGCCs was not affected. Exposure of DRG neurons to oxaliplatin (10 or 100 μM) for 24 h in vitro significantly increased the I_Ca(V) current density, with a significant influence on L- and T-type VGCCs. Immunostaining revealed an increase of L- and T-type VGCC protein levels in DRG neurons 24 h after oxaliplatin exposure. This effect was mediated by calcium-calmodulin-protein kinase II (CaMKII). Significant alterations in action potentials (AP) and their characteristics were also observed. While the amplitude increased after oxaliplatin treatment, the rise time and time-to-peak decreased, and these effects were reversed by treatment with pimozide and nimodipine, which suggests that VGCCs are critically involved in oxaliplatin-mediated neurotoxicity.     

Down-regulation of endogenous KLHL1 decreases voltage-gated calcium current density

The actin-binding protein Kelch-like 1 (KLHL1) can modulate voltage-gated calcium channels in vitro. KLHL1 interacts with actin and with the pore-forming subunits of Cav2.1 and CaV3.2 calcium channels, resulting in up-regulation of P/Q and T-type current density. Here we tested whether endogenous KLHL1 modulates voltage gated calcium currents in cultured hippocampal neurons by down-regulating the expression of KLHL1 via adenoviral delivery of shRNA targeted against KLHL1 (shKLHL1). Control adenoviruses did not affect any of the neuronal properties measured, yet down-regulation of KLHL1 resulted in HVA current densities ∼68% smaller and LVA current densities 44% smaller than uninfected controls, with a concomitant reduction in α_1A and α_1H protein levels. Biophysical analysis and western blot experiments suggest Ca_V3.1 and 3.3 currents are also present in shKLHL1-infected neurons. Synapsin I levels, miniature postsynaptic current frequency, and excitatory and inhibitory synapse number were reduced in KLHL1 knockdown. This study corroborates the physiological role of KLHL1 as a calcium channel modulator and demonstrates a novel, presynaptic role.     

TRPgamma channels are inhibited by cAMP and contribute to pacemaking in neurosecretory insect neurons

From a neuronal cDNA library of the cockroach Periplaneta americana we isolated a 3585-bp cDNA sequence encoding Periplaneta transient receptor potential gamma (pTRPgamma), a protein of 1194 amino acids showing 65% identity to the orthologous Drosophila channel protein dTRPgamma. Heterologous expression of pTRPgamma in HEK293 cells produced a constitutively active, non-selective cation channel with a Ca2+:Na+ permeability ratio of 2. In contrast to dTRPgamma-mediated currents, pTRPgamma currents were partially inhibited by 8-bromo-cAMP, and this effect was not mediated by protein kinase A (PKA) activation. pTRPgammab, a truncated pTRPgamma splice variant missing most of the C terminus, was insensitive to 8-bromo-cAMP. Thus, the critical cAMP-binding site seems to be located in the C-terminal part of pTRPgamma, although there is no common cAMP-binding consensus sequence. While dTRPgamma is only expressed in the photoreceptors, pTRPgamma is expressed throughout the nervous system. In particular it is expressed in dorsal unpaired median (DUM) neurons. In these octopamine-releasing, neurosecretory cells a Ca2+ background current contributing to pacemaker activity was found to be up-regulated by the reduction of cAMP level. In addition, the Ca2+ background current was inhibited by LOE-908, 2-APB, and La3+, which similarly affected the pTRPgamma current. We thus propose that the pTRPgamma protein is involved in forming the channel passing the Ca2+ pakemaking background current in DUM neurons.     

Mas-related G protein-coupled receptor D is coupled to endogenous calcium-activated chloride channel in Xenopus oocytes

Mas-related G protein-coupled receptor D (MrgD) is expressed almost exclusively in nociceptive primary sensory neurons and the neurons located in stratum granulosum of skin. More and more evidence suggest that MrgD plays an important role in pain sensation and/or transduction. Recent studies have demonstrated that the receptor is also involved in itch sensation in both mouse and human. In the present study, we identified a robust inward current in MrgD-expressing Xenopus oocytes by using β-alanine, a putative ligand of MrgD. The currents were sensitive to inhibitor of Ca²⁺-activated chloride channels (CaCCs) and intracellular Ca²⁺ chelator, suggesting they were produced by endogenous CaCCs. Furthermore, it was demonstrated that upon the application of phospholipase C (PLC) inhibitor, or antisense oligonucleotides of inositol trisphosphate receptor (IP3R), the β-alanine-induced currents were dramatically depressed. However, protein kinase C inhibitor did not display any visible effect on CaCC currents. In summary, our data suggest that the activation of MrgD promotes the open of endogenous CaCCs via G_q-PLC-IP3-Ca²⁺ pathway. The current findings reveal the functional coupling between MrgD and CaCCs in Xenopus oocytes and also provide a facile model to assay the activity of MrgD.     

Cellular action of cholecystokinin-8S-mediated excitatory effects in the rat periaqueductal gray

The peptide cholecystokinin (CCK) is one of the major neurotransmitters modulating satiety, nociception, and anxiety behavior. Although many behavioral studies showing anti-analgesic and anxiogenic actions of CCK have been reported, less is known about its cellular action in the central nervous system (CNS). Therefore, we examined the action of CCK in rat dorsolateral periaqueductal gray (PAG) neurons using slice preparations and whole-cell patch-clamp recordings. Application of CCK-8S produced an inward current accompanied by increased spontaneous synaptic activities. The CCK-8S-induced inward current (I(CCK)) was recovered after washout and reproduced by multiple exposures. Current-voltage plots revealed that I(CCK) reversed near the equilibrium potential for K(+) ions with a decreased membrane conductance. When several K(+) channel blockers were used, application of CdCl(2), TEA, or apamin significantly reduced I(CCK). I(CCK) was also significantly reduced by the CCK(2) receptor antagonist, L-365,260, while it was not affected by the CCK(1) receptor antagonist, L-364,718. Furthermore, we examined the effects of CCK-8S on miniature excitatory postsynaptic currents (mEPSCs) in order to determine the mechanism of CCK-mediated increase on synaptic activities. We found that CCK-8S increased the frequency of mEPSCs, but had no effect on mEPSC amplitude. This presynaptic effect persisted in the presence of CdCl(2) or Ca(2+)-free bath solution, but was completely abolished by pre-treatment with BAPTA-AM, thapsigargin or L-365,260. Taken together, our results indicate that CCK can excite PAG neurons at both pre- and postsynaptic loci via the activation of CCK(2) receptors. These effects may be important for the effects of CCK on behavior and autonomic function that are mediated via PAG neurons.     

Voltage-dependent and Src-mediated regulation of NMDA receptor single channel outward currents in cortical neurons

A voltage-dependent but Ca²⁺-independent regulation of N-methyl-D-aspartate (NMDA) receptor outward activity was studied at the single channel level using outside-out patches of cultured mouse cortical neurons. Unlike the inward activity associated with Ca²⁺ and Na⁺ influx, the NMDA receptor outward K⁺ conductance was unaffected by changes in Ca²⁺ concentration. Following a depolarizing pre-pulse, the single channel open probability (NP _o), amplitude, and open duration of the NMDA inward current decreased, whereas the same pre-depolarization increased those parameters of the NMDA outward current (pre-pulse facilitation). The outward NP _o was increased by the pre-pulse facilitation, disregarding Ca²⁺ changes. The voltage–current relationships of the inward and outward currents were shifted by the pre-depolarization toward opposite directions. The Src family kinase inhibitor, PP1, and the Src kinase antibody, but not the anti-Fyn antibody, blocked the pre-pulse facilitation of the NMDA outward activity. On the other hand, a hyperpolarizing pre-pulse showed no effect on NMDA inward currents but inhibited outward currents (pre-pulse depression). Application of Src kinase, but not Fyn kinase, prevented the pre-pulse depression. We additionally showed that a depolarization pre-pulse potentiated miniature excitatory synaptic currents (mEPSCs). The effect was blocked by application of the NMDA receptor antagonist AP-5 during depolarization. These data suggest a voltage-sensitive regulation of NMDA receptor channels mediated by Src kinase. The selective changes in the NMDA receptor-mediated K⁺ efflux may represent a physiological and pathophysiological plasticity at the receptor level in response to dynamic changes in the membrane potential of central neurons.     

β-Adrenergic Modulation of Glial Inwardly Rectifying Potassium Channels

Abstract: Cultured spinal cord astrocytes (2–13 days in vitro) express several different potassium current types, including delayed rectifier, transient A-type, and inward rectifier (K_ir) K⁺ currents. Of these, K_ir is believed to be of critical importance in the modulation of extracellular [K⁺] in the CNS. Using the whole-cell patch-clamp technique, we analyzed modulation of K_ir currents by β-adrenergic receptor activation. The selective β-adrenergic agonist isoproterenol (1–100 µM) and epinephrine (1–100 µM) each reduced peak K_ir current amplitudes to 52.7 ± 12.5 and 63.6 ± 7.0%, respectively, at 100 µM. Forskolin (K_D of ～25 µM), an activator of adenylate cyclase (AC), and dibutyryl-cyclic AMP (1 mM), a membrane-permeable analogue of cyclic AMP (cAMP), were each used to increase [cAMP]_i, the product of AC, and resulted in similar reductions of K_ir currents. By contrast, 1,9-dideoxyforskolin (1–50 µM), a forskolin analogue that does not activate AC, did not affect K_ir currents, indicating that AC activity is a required element for K_ir modulation. Three inhibitors of PKA—Rp-adenosine 3′,5′-cyclic monophosphothioate, H-7, and adenosine 3′,5′-cyclic monophosphate-dependent protein kinase inhibitor—failed to inhibit K_ir current reduction by β-adrenergic agonists. These results indicate that β-adrenergic receptor ligands can modulate K_ir currents and suggest that this modulation involves activation of AC but not protein kinase A. Such modulation may provide a mechanism by which neurons can modulate glial K_ir currents and thereby may affect glial K⁺“spatial buffering” in the CNS.     

Protein kinase C enhances plasma membrane expression of cardiac L-type calcium channel,CaV1.2

L-type-voltage-dependent Ca²⁺ channels (L-VDCCs; Ca_V1.2, α_1C), crucial in cardiovascular physiology and pathology, are modulated via activation of G-protein-coupled receptors and subsequently protein kinase C (PKC). Despite extensive study, key aspects of the mechanisms leading to PKC-induced Ca²⁺ current increase are unresolved. A notable residue, Ser1928, located in the distal C-terminus (dCT) of α_1C was shown to be phosphorylated by PKC. Ca_V1.2 undergoes posttranslational modifications yielding full-length and proteolytically cleaved CT-truncated forms. We have previously shown that, in Xenopus oocytes, activation of PKC enhances α_1C macroscopic currents. This increase depended on the isoform of α_1C expressed. Only isoforms containing the cardiac, long N-terminus (L-NT), were upregulated by PKC. Ser1928 was also crucial for the full effect of PKC. Here we report that, in Xenopus oocytes, following PKC activation the amount of α_1C protein expressed in the plasma membrane (PM) increases within minutes. The increase in PM content is greater with full-length α_1C than in dCT-truncated α_1C, and requires Ser1928. The same was observed in HL-1 cells, a mouse atrium cell line natively expressing cardiac α_1C, which undergoes the proteolytic cleavage of the dCT, thus providing a native setting for exploring the effects of PKC in cardiomyocytes. Interestingly, activation of PKC preferentially increased the PM levels of full-length, L-NT α_1C. Our findings suggest that part of PKC regulation of Ca_V1.2 in the heart involves changes in channel's cellular fate. The mechanism of this PKC regulation appears to involve the C-terminus of α_1C, possibly corroborating the previously proposed role of NT-CT interactions within α_1C.     

The effects of Al on the calcium currents inHelix neurons

Summary 1. The effects of aluminium (Al) on calcium (Ca) currents were investigated by using the conventional two-electrode voltage clamp technique inHelix pomatia neurons. The peak amplitude, kinetics, and voltage dependence of activation and inactivation of the Ca currents were studied in the presence of 10^–5–10^–3 M AlCl₃, at pH 6.2. Al prolonged the rising phase of the Ca currents and therefore increased the time to peak at each command voltage step used.3. There was no significant influence of Al on the peak amplitude of the Ca currents, but the voltage dependence of the time to peak, activation, and inactivation of the Ca currents shifted to more positive potentials as a consequence of Al treatment.4. The leak currents were not influenced by Al up to 1 mM, which was the maximal dose applied.5. The results support the suggestion that Al may modify the Ca homeostasis and that it exerts a neurotoxic effect, at least in part, by modulation of the Ca current of the neuronal membrane.     

Effects of Estradiol on Voltage-Gated Potassium Channels in Mouse Dorsal Root Ganglion Neurons

Voltage-gated potassium channels are regulators of membrane potentials, action potential shape, firing adaptation, and neuronal excitability in excitable tissues including in the primary sensory neurons of dorsal root ganglion (DRG). In this study, using the whole-cell patch-clamp technique, the effect of estradiol (E2) on voltage-gated total outward potassium currents, the component currents transient “A-type” current (I _A) currents, and “delayed rectifier type” (I _KDR) currents in isolated mouse DRG neurons was examined. We found that the extracellularly applied 17β-E2 inhibited voltage-gated total outward potassium currents; the effects were rapid, reversible, and concentration-dependent. Moreover, the membrane impermeable E2-BSA was as efficacious as 17β-E2, whereas 17α-E2 had no effect. 17β-E2-stimulated decrease in the potassium current was unaffected by treatment with ICI 182780 (classic estrogen receptor antagonist), actinomycin D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor). We also found that I _A and I _KDR were decreased after 17β-E2 application. 17β-E2 significantly shifted the activation curve for I _A and I _KDR channels in the hyperpolarizing direction. In conclusion, our results demonstrate that E2 inhibited voltage-gated K⁺ channels in mouse DRG neurons through a membrane ER-activated non-genomic pathway.     

Mechanisms of the inhibitory effect of cyclic adenosine monophosphate on calcium current in intact mollusk neurons

Inhibitory effects of cyclic adenosine monophosphate (cAMP) on calcium current (I_Ca) were investigated in experiments on unidentified neurons isolated fromHelix pomatia by means of voltage clamping techniques using two microelectrodes. Intracellular level of cAMP was raised by intracellular injection of this substance or by extracellular application of dibutyryl-cAMP or isobutylmethyl-xanthine. A set of neurons showing inhibitory effects of cAMP on I_Ca was used. Effects on barium current (I_Ba) of an equal extent were also revealed. Injection of cGMP through a double-barreled microelectrode into these neurons produced an increase in amplitude of I_Ca. Intracellular application of phorbol ester had no effect on this current, however. Intracellular injection of EGTA led to enhancement of I_Ca amplitude, but the inhibitory effect of cAMP persisted following the action of EGTA. Tolbutamide and H-8 (but to a lesser extent) inhibited I_Ca. The inhibitory effects of tolbutamide and dibutyryl cAMP were not found to be cumulative in six out of twelve instances. These findings would imply that the inhibitory action of cAMP on I_Ca is unassociated with activation of cAMP-dependent protein kinase, cGMP-dependent protein kinase or protein kinase C; nor does it depend on level of intracellular Ca²⁺. The possibility of direct interaction between cAMP and channel-forming protein is considered.Institute of Brain Research, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 54–61, January–February, 1990.