Channel Currents In Neurons Research Articles

Positive Allosteric Modulation of the 5-HT1A Receptor by Indole-Based Synthetic Cannabinoids Abused by Humans.

The nonmedical (i.e., recreational) misuse of synthetic cannabinoids (SCs) is a worldwide public health problem. When compared to cannabis, the misuse of SCs is associated with a higher incidence of serious adverse effects, suggesting the possible involvement of noncannabinoid sites of action. Here, we find that, unlike the phytocannabinoid Δ9-tetrahydrocannabinol, the indole-moiety containing SCs, AM2201 and JWH-018, act as positive allosteric modulators (PAMs) at the 5-HT1A receptor (5-HT1AR). This suggests that some biological effects of SCs might involve allosteric interactions with 5-HT1ARs. To test this hypothesis, we examined effects of AM2201 on 5-HT1AR agonist-activated G protein-coupled inwardly rectifying potassium channel currents in neurons in vitro and on the hypothermic response to 5-HT1AR stimulation in mice lacking the cannabinoid receptor 1. We found that both 5-HT1AR effects were potentiated by AM2201, suggesting that PAM activity at 5-HT1AR may represent a novel noncannabinoid receptor mechanism underlying the complex profile of effects for certain SCs.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Currents in Neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that activate at potentials more negative than -50 mV and are predominantly permeable to Na(+) and K(+) ions. Four HCN subunits (HCN1-4) have been cloned. These subunits have distinct expression patterns and biophysical properties. In addition, cyclic nucleotides as well as multiple intracellular substances including various kinases and phosphatases modulate the expression and function of the subunits. Hence, the characteristics of the current, Ih, are likely to vary among neuronal subtypes. In many neuronal subtypes, Ih is present postsynaptically, where it plays a critical role in setting the resting membrane potential and the membrane resistance. By influencing these intrinsic properties, Ih will affect synaptic potential shapes and summation and thereby affect neuronal excitability. Additionally, Ih can have an effect on resonance properties and intrinsic neuronal oscillations. In some neurons, Ih may also be present presynaptically in axons and synaptic terminals, where it modulates neuronal transmitter release. Hence the effects of Ih on neuronal excitability are complex. It is, however, necessary to fully understand these as Ih has a significant impact on physiological conditions such as learning as well as pathophysiological states such as epilepsy.

Curcumin Protects Microglia and Primary Rat Cortical Neurons against HIV-1 gp120-Mediated Inflammation and Apoptosis

Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9) and primary rat cortical neurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop) protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K+) current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K+ channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the delayed rectification and transient outward K+ channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects cortical neurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K+ current.

Properties of BK-type Ca++-dependent K+ channel currents in medial prefrontal cortex pyramidal neurons in rats of different ages

The medial prefrontal cortex (PFC) is involved in cognitive functions, which undergo profound changes during adolescence. This alteration of the PFC function derives from neuron activity, which, in turn, may depend on age-dependent properties and the expression of neuronal ion channels. BK-type channels are involved in controlling both the Ca++ ion concentration in the cell interior and cell excitability. The purpose of this study was to test the properties of BK currents in the medial PFC pyramidal neurons of young (18- to 22-day-old), adolescent (38- to 42-day-old), and adult (60- to 65-day-old) rats. Whole-cell currents evoked by depolarizing voltage steps were recorded from dispersed medial PFC pyramidal neurons. A selective BK channel blocker – paxilline (10 μM) – irreversibly decreased the non-inactivating K+ current in neurons that were isolated from the young and adult rats. This current was not significantly affected by paxilline in the neurons obtained from adolescent rats. The properties of single-channel K+ currents were recorded from the soma of dispersed medial PFC pyramidal neurons in the cell-attached configuration. Of the K+ channel currents that were recorded, ~90% were BK and leak channel currents. The BK-type channel currents were dependent on the Ca++ concentration and the voltage and were inhibited by paxilline. The biophysical properties of the BK channel currents did not differ among the pyramidal neurons isolated from young, adolescent, and adult rats. Among all of the recorded K+ channel currents, 38.9, 12.7, and 21.1% were BK-type channel currents in the neurons isolated from the young, adolescent, and adult rats, respectively. Furthermore, application of paxilline effectively prolonged the half-width of the action potential in pyramidal neurons in slices isolated from young and adult rats but not in neurons isolated from adolescent rats. We conclude that the availability of BK channel currents decreases in medial PFC pyramidal neurons of adolescent rats compared with those in the neurons of young and adult rats while their properties did not change across ages.

The κ-Opioid Receptor Agonist U-50488 Blocks Ca2+ Channels in a Voltage- and G Protein–Independent Manner in Sensory Neurons

κ-Opioid receptor (κ-OR) activation is known to play a role in analgesia and central sedation. The purpose of the present study was to examine the effect of the κ-OR agonist, U-50488 (an arylacetamide), on Ca channel currents and the signaling proteins involved in acutely isolated rat dorsal root ganglion (DRG) neurons expressing the putative promoter region of the tetrodotoxin-resistant Na channel (NaV 1.8) that is known to be involved in pain transmission. Acutely isolated rat DRG neurons were transfected with cDNA coding for enhanced green fluorescent protein (EGFP), whose expression is driven by the NaV 1.8 promoter region. Thereafter, the whole-cell variant of the patch-clamp technique was used to record Ca channel currents in neurons expressing EGFP. Exposure of EGFP-expressing DRG neurons to U-50488 (0.3-40 μM) led to voltage-independent inhibition of the Ca channel currents. The modulation of the Ca currents did not appear to be mediated by the Gα protein subfamilies: Gαi/o, Gαs, Gαq/11, Gα14, and Gαz. Furthermore, dialysis of the hydrolysis-resistant GDP analog, GDP-β-S (1 mM), did not affect the U-50488-mediated blocking effect, ruling out involvement of other G protein subunits. Finally, U-50488 (20 μM) blocked Ca channels heterologously expressed in HeLa cells that do not express κ-OR. These results suggest that the antinociceptive actions mediated by U-50488 are likely due to both a direct block of Ca channels in sensory neurons as well as G protein modulation of Ca currents via κ-OR-expressing neurons.

Modulation of ‘A’-type K+ current by rodent and human forms of amyloid β protein

The Alzheimer's disease related peptide amyloid beta (Abeta) might have a physiological role in upregulating K channel currents in neurones. Earlier studies used the human form of Abeta1-40 on rat neurones. We sought to confirm our hypothesis by use of rat Abeta, which has no Alzheimer's association. In rat cerebellar granule neurones and HEK293 cells expressing Kv4.2 subunits, whole-cell patch clamp of K currents revealed that preincubation of cells with recombinant human or rat Abeta1-40 (10 nM for 24 h) significantly increased K channel current density. This was accompanied by increased mRNA levels for Kv4.2. These data indicate that rodent and human Abeta are effective in modulating K currents. The effectiveness of nonaggregating rat Abeta also strongly supports a physiological role for the peptide.

Pain Perception in Mice Lacking the β3 Subunit of Voltage-activated Calcium Channels

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.

Histamine modulates high-voltage-activated calcium channels in neurons dissociated from the rat tuberomammillary nucleus

The effects of histamine on high-voltage-activated Ca 2+ channels in the histaminergic neurons acutely dissociated from the rat tuberomammillary nucleus were investigated in the nystatin-perforated patch recording mode under voltage-clamp conditions. Histamine suppressed the high-voltage-activated Ca 2+ channel currents in neurons which were positive for histidine decarboxylase with immunocytochemistry. The half-maximum inhibitory concentration and maximum inhibition were 2.6×10 −7 M and 16.6±1.9%, respectively. An H 3 receptor agonist, R(−)-α-methylhistamine, mimicked the response to histamine, and thioperamide, an H 3 receptor antagonist, inhibited the response to histamine. On the other hand, neither 2-methylhistamine, an H 1 receptor agonist, nor dimaprit, an H 2 receptor agonist, had a significant effect on the Ca 2+ channel currents. Pretreatment with pertussis toxin blocked the inhibitory effect of histamine on Ca 2+ channels, suggesting the involvement of Gi/Go proteins in the action of histamine. ω-Conotoxin-GVIA, ω-agatoxin-IVA, nicardipine, and ω-conotoxin-MVIIC blocked the high-voltage-activated Ca 2+ channel currents by 15.6, 4.3, 27.1, and 31.2% of the total current, respectively, suggesting the existence of N-, P-, L-, and Q-type Ca 2+ channels. A current that was insensitive to these blockers was also found. This residual current, “R-type”, was completely suppressed by the addition of 200 μM Cd 2+. Histamine significantly inhibited both the N- and P-type current components among these five types of Ca 2+ channel currents. We concluded that histamine suppresses the N- and P-type Ca 2+ channels in histaminergic neurons through an H 3 receptor which is linked to a pertussis toxin-sensitive G-protein.

Voltage-dependent potentiation of neuronal L-type calcium channels due to state-dependent phosphorylation.

Modulation of Ca2+ channels during repetitive activity in excitable cells can have an important role in altering cellular function. In mammalian parasympathetic and dorsal root ganglion neurons, L-type Ca2+ channels are potentiated by single depolarizing prepulses or trains of short high-frequency depolarizing pulses. This type of potentiation takes place regardless of whether Ca2+ or Ba2+ is the charge carrier and requires phosphorylation by a adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The magnitude of facilitation was correlated with frequency of conditioning trains, was enhanced by 8-bromoadenosine 3',5'-cyclic monophosphate or the Sp diastereomer of adenosine 3',5'-cyclic monophosphothioate (cAMPS), and reduced by Rp-cAMPS or a peptide inhibitor of cAMP-dependent protein kinase. The N-type Ca2+ channels exhibited the opposite response to these agents. We propose that the potentiation of L-type Ca2+ channel currents in neurons is due to state-dependent phosphorylation by cAMP-dependent protein kinase (Sculptoreanu, A., T. Scheuer, and W. A. Catterall. Nature Lond. 364: 240-243, 1993; Sculptoreanu, A., E. Rotman, M. Takahashi, T. Scheuer, and W. A. Catterall. Proc. Natl. Acad. Sci. USA 90: 10135-10139, 1993.). Thus state-dependent phosphorylation in neurons may be a mechanism for the regulation of various functions including transmitter release.

P-type calcium channels in rat central and peripheral neurons

The peptide toxin ω-Aga-IVA blocked P-type Ca 2+ channel current in rat Purkinje neurons (K D ≈ 2 nM) but had no effect on identified T-type, L-type, or N-type currents in a variety of central and peripheral neurons. ω-Aga-IVA blocked a substantial fraction of high threshold Ca 2+ channel current in neurons from the hippocampal CA1 region (mean 26%), visual cortex (32%), spinal cord (45%), and dorsal root ganglia (23%), but less in hippocampal CA3 neurons (14%) and none in sympathetic neurons. In all cases, ω-Aga-IVA block could be reversed by a brief train of strong depolarizations. There was no overlap between current blocked by ω-Aga-IVA and the fractions blocked by dihydropyridines and ω-conotoxin GVIA, but not all current resistant to dihydropyridines and ω-conotoxin was blocked by ω-Aga-IVA. The results suggest that ω-Aga-IVA is highly selective for P-type channels and that many central neurons and some peripheral neurons possess substantial P-type current.

Characterization of single non-inactivating potassium channels in primary neuronal cultures of Drosophila

Permeability and gating properties of single, non-inactivating, K+ channel currents in cultured Drosophila neurons were studied using the gigaohm-seal patch-clamp technique. The non-inactivating K+ currents were activated by depolarizing the membrane to -30 mV or to more positive potentials. The slope conductance of the channel was estimated to be 17.6 +/- 3.70 pS when the cytoplasmic side of the inside-out membrane patch was perfused with solutions containing 145 mmoll-1 K+. The single-channel conductance was temperature-sensitive, with a Q10 of 1.44 between 10 and 20 degrees C. Single-channel currents could be recorded when the cytoplasmic K+ was replaced with NH4+, Rb+ or Na+, but not with Cs+. The conductance ratio of the channel for these cations was: K+ (1) greater than NH4+(0.53) greater than Rb+ (0.47) greater than Na+ (0.44). Tetraethylammonium (TEA+) ions applied at a concentration of 10 mmoll-1 to the cytoplasmic side of the membrane increased the frequency of 'blank' traces which contained no channel openings during repetitive depolarization. In addition, single-channel amplitude was reduced by about 20%. The open-time distribution was fitted by a single exponential function, whereas the closed-time distribution required a three-exponential fit. Permeability and gating properties of single, non-inactivating K+ channel currents in neurons of eag, a mutant which has defects in the delayed rectifier K+ channel, were indistinguishable from those recorded from wild-type neurons.