Molecular cloning, functional characterization and differential expression of two novel GABAAR-like subunits from red swamp crayfish Procambarus clarkii.

In this work, we cloned and functionally expressed two novel GABAA receptor subunits from Procambarus clarkii crayfish. These two new subunits, PcGABAA-α and PcGABAA-ß2, revealed significant sequence homology with the PcGABAA-ß subunit, previously identified in our laboratory. In addition, PcGABAA-α subunit also shared a significant degree of identity with the Drosophila melanogaster genes DmGRD (GABA and glycine-like receptor subunits of Drosophila) as well as PcGABAA-ß2 subunit with DmLCCH3 (ligand-gated chloride channel homolog 3). Electrophysiological recordings showed that the expression in HEK cells of the novel subunits, either alone or in combination, failed to form functional homo- or heteromeric receptors. However, the co-expression of PcGABAA-α with PcGABAA-ß evoked sodium- or chloride-dependent currents that accurately reproduced the time course of the GABA-evoked currents in the X-organ neurons from crayfish, suggesting that these GABA subunits combine to form two types of GABA receptors, one with cationic selectivity filter and the other preferentially permeates anions. On the other hand, PcGABAA-ß2 and PcGABAA-ß co-expression generated a chloride current that does not show desensitization. Muscimol reproduced the time course of GABA-evoked currents in all functional receptors, and picrotoxin blocked these currents; bicuculline did not block any of the recorded currents. Reverse transcription polymerae chain reaction (RT-PCR) amplifications and FISH revealed that PcGABAA-α and PcGABAA-ß2 are predominantly expressed in the crayfish nervous system. Altogether, these findings provide the first evidence of a neural GABA-gated cationic channel in the crayfish, increasing our understanding of the role of these new GABAA receptor subunits in native heteromeric receptors.

Synthetic Monopartite Peptide That Enables the Nuclear Import of Genes Delivered by the Neurotensin-Polyplex Vector.

Neurotensin (NTS)-polyplex is a multicomponent nonviral vector that enables gene delivery via internalization of the neurotensin type 1 receptor (NTSR1) to dopaminergic neurons and cancer cells. An approach to improving its therapeutic safety is replacing the viral karyophilic component (peptide KPSV40; MAPTKRKGSCPGAAPNKPK), which performs the nuclear import activity, by a shorter synthetic peptide (KPRa; KMAPKKRK). We explored this issue and the mechanism of plasmid DNA translocation through the expression of the green fluorescent protein or red fluorescent protein fused with KPRa and internalization assays and whole-cell patch-clamp configuration experiments in a single cell together with importin α/ß pathway blockers. We showed that KPRa electrostatically bound to plasmid DNA increased the transgene expression compared with KPSV40 and enabled nuclear translocation of KPRa-fused red fluorescent proteins and plasmid DNA. Such translocation was blocked with ivermectin or mifepristone, suggesting importin α/ß pathway mediation. KPRa also enabled NTS-polyplex-mediated expression of reporter or physiological genes such as human mesencephalic-derived neurotrophic factor (hMANF) in dopaminergic neurons in vivo. KPRa is a synthetic monopartite peptide that showed nuclear import activity in NTS-polyplex vector-mediated gene delivery. KPRa could also improve the transfection of other nonviral vectors used in gene therapy.

Presynaptic cannabinoid CB2 receptors modulate [3 H]-Glutamate release at subthalamo-nigral terminals of the rat.

Recent studies suggested the expression of CB2 receptors in neurons of the CNS, however, most of these studies have only explored one aspect of the receptors, i.e., expression of protein, messenger RNA, or functional response, and more complete studies appear to be needed to establish adequately their role in the neuronal function. Electron microscopy studies showed the presence of CB2r in asymmetric terminals of the substantia nigra pars reticulata (SNr), and its mRNA appeared is expressed in the subthalamic nucleus. Here, we explore the expression, source, and functional effects of such receptors by different experimental approaches. Through PCR and immunochemistry, we showed mRNA and protein for CB2rs in slices and primary neuronal cultures from subthalamus. GW833972A, GW405833, and JHW 133, three CB2r agonists dose-dependent inhibited K+ -induced [3 H]-Glutamate release in slices of SNr, and the two antagonist/inverse agonists, JTE-907 and AM630, but not AM281, a CB1r antagonist, prevented GW833972A effect. Subthalamus lesions with kainic acid prevented GW833972A inhibition on release and decreased CB2r protein in nigral synaptosomes, thus nigral CB2rs originate in subthalamus. Inhibition of [3 H]-Glutamate release was PTX- and gallein-sensitive, suggesting a Gißγ -mediated effect. P/Q Ca2+ -type channel blocker, ω-Agatoxin-TK, also inhibited the [3 H]-Glutamate release, this effect was occluded with GW833972A inhibition, indicating that the ßγ subunit effect is exerted on Ca2+ channel activity. Finally, microinjections of GW833972A in SNr induced contralateral turning. Our data showed that presynaptic CB2rs inhibit [3 H]-Glutamate release in subthalamo-nigral terminals by P/Q-channels modulation through the Gißγ subunit and suggested their participation in motor behavior.

Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA ß subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons.

In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones.

Inhibition of SERCA pumps induces desynchronized RyR activation in overloaded internal Ca2+ stores in smooth muscle cells.

We have previously shown that rapid inhibition of sarcoplasmic reticulum (SR) ATPase (SERCA pumps) decreases the amplitude and rate of rise (synchronization) of caffeine induced-Ca(2+) release without producing a reduction of free luminal SR Ca(2+) level in smooth muscle cells (Gómez-Viquez L, Guerrero-Serna G, García U, Guerrero-Hernández A. Biophys J 85: 370-380, 2003). Our aim was to investigate the role of luminal SR Ca(2+) content in the communication between ryanodine receptors (RyRs) and SERCA pumps. To this end, we studied the effect of SERCA pump inhibition on RyR-mediated Ca(2+) release in smooth muscle cells with overloaded SR Ca(2+) stores. Under this condition, the amplitude of RyR-mediated Ca(2+) release was not affected but the rate of rise was still decreased. In addition, the caffeine-induced Ca(2+)-dependent K(+) outward currents revealed individual events, suggesting that SERCA pump inhibition reduces the coordinated activation of RyRs. Collectively, our results indicate that SERCA pumps facilitate the activation of RyRs by a mechanism that does not involve the regulation of SR Ca(2+) content. Importantly, SERCA pumps and RyRs colocalize in smooth muscle cells, suggesting a possible local communication between these two proteins.

An improved method for long-term measuring of hemolymph fluctuations of non-essential amino acids, GABA and histamine from freely moving crayfish.

The microdialysis method was adapted to obtain long-term hemolymph dialysates from the pericardial cavity of freely moving Procambarus clarkii crayfish, to measure fluctuations of non-essential amino acids, GABA and histamine by high-performance liquid chromatography using off-line fluorometric derivatization. Asp, Ala, Tau, GABA and histamine (HA) reached its maximal concentrations at the daybreak, whereas glutamate (Glu), Gln and Gly peaked at the end of the light period. The minimum and maximal detected amounts for each substance along the 24h cycle were (in microM): 20-300Asp, 100-200Glu, 400-700Gln, 400-600Gly, 100-200Tau, 150-300Ala, 2-10 GABA and 25-250HA. Cocktails containing the relative concentration of each amino acid, GABA and histamine resulted in a hyperpolarization that reduced the spontaneous firing of cultured peptidergic X organ neurons. Glu, GABA and histamine evoked a long-lasting hyperpolarization that suppressed the spontaneous firing, whereas Asp, Gly and Tau evoked a depolarization accompanied with neuronal firing. Finally, neither Ala nor Gln modified the resting membrane potential.

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells.

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.

Expression and functional characterization of GABA transporters in crayfish neurosecretory cells.

The effect of GABA on membrane potential and ionic currents of X-organ neurons isolated from the crayfish eyestalk was investigated. Under voltage-clamp conditions, GABA elicited an inward Na+ current followed by a sustained outward chloride current. Sodium current was partially blocked in a dose-dependent manner by antagonists of GABA plasma membrane transporters such as beta-alanine, nipecotic acid, 1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711), and SKF89976-A at concentrations between 1 and 100 microm. This current was totally blocked by the combined application of NO 711 (5 microm) and beta-alanine (50 microm). We obtained an EC(50) of 5 microm and a Hill coefficient of 0.97 for the GABA transport mediated response. These results together with studies of immunolocalization using antibodies against neuronal vertebrate GABA transporters (GATs) indicate the presence of GAT-1- and GAT-3-like proteins in X-organ neurons. To isolate the sustained outward Cl- current, extracellular free sodium solution was used to minimize the contribution of GAT activity. We concluded that this current was caused by the activation of GABA(A)-like receptors with an EC50 of 10 microm and a Hill number of 1.7. To assign a functional role to the GATs in the X-organ sinus gland system, we determine the GABA concentration (0.46-0.15 microm) in hemolymph samples using HPLC. In summary, our results suggest that a sodium-dependent electrogenic GABA uptake mechanism has a direct influence on the excitability of the X-organ neurons, maintaining an excitatory tone that is dependent on the circulating GABA level.

Histamine operates Cl -gated channels in crayfish neurosecretory cells.

We describe a histamine-activated Cl(-) conductance in the X-organ neurons from crayfish Cherax quadricarinatus, which has comparable properties to the homomultimeric histamine-gated ion channels described in Drosophila. Topical application of histamine inhibited spontaneous neuronal firing in the X-organ sinus gland tract, concomitant with an increase in the membrane conductance. In X-organ neurons in culture and under voltage-clamp conditions, histamine evoked outward currents at -40 mV that reversed at the Cl(-) equilibrium potential. Histamine sensitivity in these neurons had a half-maximal response (EC(50))=3.3+/-1 micromol l(-1), with a Hill number of 2.6+/-0.4. The histamine-evoked current was blocked by tiotidine, cimetidine, ranitidine and 256+/-11 and 483+/-11 micromol l(-1), respectively) and d-tubocurarine (IC(50)=21+/-2 micromol l(-1)), but was insensitive to picrotoxin, bicuculline and strychnine. Neither GABA nor glutamate was capable of desensitizing the histamine response, indicating that histamine activates a particular Cl(-) conductance. The presence of immunoreactive neurons to histamine in the medulla terminalis with axonal projections to the neuropile suggests a possible histaminergic modulation of the X-organ sinus gland system.

SERCA pump optimizes Ca2+ release by a mechanism independent of store filling in smooth muscle cells.

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores.

Cn11, the first example of a scorpion toxin that is a true blocker of Na(+) currents in crayfish neurons.

A novel crustacean toxin (Cn11) was isolated and characterized from the venom of the Mexican scorpion Centruroides noxius Hoffmann. It contains 63 amino acid residues and is stabilized by four disulphide bridges. It is lethal to crustaceans (Cambarellus montezumae), less toxic to insects (crickets) and non-toxic to mammals (mice) at the doses assayed. In neurons isolated from the X organ-sinus gland system of the crayfish Procambarus clarkii, it blocks the Na(+) currents with an estimated K(m) of 320 nmol l(-1), without affecting the Ca(2+) and K(+) currents. The voltage-gated tetrodotoxin-sensitive Na(+) current was recorded from X organ neurons in culture 24 h after plating using the whole-cell clamp configuration. The Na(+) current was isolated by blocking Ca(2+) currents with Cd(2+) and Cs(+) and K(+) currents with tetraethylammonium and 4-aminopyridine. Under control conditions, the Na(+) currents were activated at -40 mV with a maximum amplitude at 0 mV. In the presence of 1 micromol l(-1) Cn11, the Na(+) current amplitude was reduced by 75 % without apparent modifications to the gating mechanism. These findings suggest that Cn11 selectively blocks a Na(+) channel. It is the first representative of a new group of scorpion toxins specific for this molecular target.