GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.

GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

An ERG channel inhibitor from the scorpion Buthus eupeus.

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.

Inhibition of potassium and calcium currents in neurones by molecularly-defined P2Y receptors.

Messenger RNAs and cDNAs for individual cloned P2Y(1), P2Y2 and P2Y(6) nucleotide receptors have been expressed by micro-injection into dissociated rat superior cervical sympathetic neurones and the effects of stimulating the expressed receptors on voltage-activated N-type Ca(2+) currents and M-type K(+) currents recorded. Both currents were reduced by stimulating all three receptors, with the following mean IC(50) values: P2Y(1) (agonist: ADP) - I(K(M)) 6.9 nM, I(Ca) 8.2 nM; P2Y(2) (agonist: UTP) - I(K(M)) 1.5 microM, I(Ca) 0.5 microM; P2Y(6) (agonist: UDP) - I(K(M)) 30 nM, I(Ca) 5.9 nM. Inhibition of I(K(M)) was voltage-independent and insensitive to Pertussis toxin; inhibition of I(Ca) showed both voltage-sensitive and insensitive, and Pertussis toxin-sensitive and insensitive components. It is concluded that these P2Y receptors can couple to more than one G protein and thereby modulate more than one ion channel. It is suggested that these effects on K(M) and Ca(N) channels may induce both postsynaptic excitory and presynaptic inhibitory responses.

The P2Y(1) receptor closes the N-type Ca(2+) channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists.

The rat P2Y(1) nucleotide receptor, the P2Y subtype abundant in the brain, was heterologously expressed in rat superior cervical ganglion neurones by micro-injection of the receptor cRNA or cDNA. ADP inhibited the N-type Ca(2+) current by 64%, with EC(50) 8.2 nM, an action blocked competitively by the P2Y(1) receptor antagonist adenosine 3', 5'-bis-phosphate (K(i) 0.7 microM). 2-Methylthio-ADP inhibited the Ca(2+) current likewise, but with EC(50) 0.57 nM, giving the highest potency reported therewith for P2Y(1). Significantly, ATP and 2-methylthio-ATP were also agonists, the latter again at a very high potency (EC(50) 2.5 nM). We propose that this neuronal receptor, when present in brain at a high density as at synapses, can respond to very low concentrations of ATP and ADP as agonists, and that this would result in inhibition of N-type Ca(2+) currents and hence can reduce transmitter release or increase neuronal excitability.

Heteromeric assembly of GABA(B)R1 and GABA(B)R2 receptor subunits inhibits Ca(2+) current in sympathetic neurons.

Neuronal GABA(B) receptors regulate calcium and potassium currents via G-protein-coupled mechanisms and play a critical role in long-term inhibition of synaptic transmission in the CNS. Recent studies have demonstrated that assembly of GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits into functional heterodimers is required for coupling to potassium channels in heterologous systems. However whether heterodimerization is required for the coupling of GABA(B) receptors to effector systems in neurons remains to be established. To address this issue, we have studied the coupling of recombinant GABA(B) receptors to endogenous Ca(2+) channels in superior cervical ganglion (SCG) neurons using nuclear microinjection to introduce both sense and antisense expression constructs. Patch-clamp recording from neurons injected with both GABA(B)R1a/1b and GABA(B)R2 cDNAs or with GABA(B)R2 alone produced marked baclofen-mediated inhibition of Ca(2+) channel currents via a pertussis toxin-sensitive mechanism. The actions of baclofen were blocked by CGP62349, a specific GABA(B) antagonist, and were voltage dependent. Interestingly, SCGs were found to express abundantly GABA(B)R1 but not GABA(B)R2 at the protein level. To determine whether heterodimerization of GABA(B)R1 and GABA(B)R2 subunits was required for Ca(2+) inhibition, the GABA(B)R2 expression construct was microinjected with a GABA(B)R1 antisense construct. This resulted in a dramatic decrease in the levels of the endogenous GABA(B)R1 protein and a marked reduction in the inhibitory effects of baclofen on Ca(2+) currents. Therefore our results suggest that in neurons heteromeric assemblies of GABA(B)R1 and GABA(B)R2 are essential to mediate GABAergic inhibition of Ca(2+) channel currents.

Dual coupling of heterologously-expressed rat P2Y6 nucleotide receptors to N-type Ca2+ and M-type K+ currents in rat sympathetic neurones.

1. The P2Y6 receptor is a uridine nucleotide-specific G protein-linked receptor previously reported to stimulate the phosphoinositide (PI) pathway. We have investigated its effect in neurones, by micro-injecting its cRNA into dissociated rat sympathetic neurones and recording responses of N-type Ca2+ (I(Ca(N))) and M-type K+ (I(K(M))) currents. 2. In P2Y6 cRNA-injected neurones, UDP or UTP produced a voltage-dependent inhibition of I(Ca(N)) by approximately 53% in whole-cell (disrupted-patch) mode and by 73% in perforated-patch mode; no inhibition occurred in control cells. Mean IC50 values (whole-cell) were: UDP, 5.9+/-0.3 nM; UTP, 20+/-1 nM. ATP and ADP (1 microM) had no significant effect. Pertussis toxin (PTX) substantially (approximately 60%) reduced UTP-mediated inhibition in disrupted patch mode but not in perforated-patch mode. 3. Uridine nucleotides also inhibited I(K(M)) in P2Y6 cRNA-injected cells (by up to 71% at 10 microM UTP; perforated-patch). Mean IC50 values were: UDP, 30+/-3 nM; UTP, 115+/-12 nM. ATP (10 microM) again had no effect. No significant inhibition occurred in control cells. Inhibition was PTX-resistant. 4. Thus, the P2Y6 receptor, like the P2Y2 subtype studied in this system, couples to both of these two neuronal ion channels through at least two different G proteins. However, the P2Y6 receptor displays a much higher sensitivity to its agonists than the P2Y2 receptor in this expression system and higher than previously reported using other expression methods. The very high sensitivity to both UDP and UTP suggests that it might be preferentially activated by any locally released uridine nucleotides.

Intracellular retention of recombinant GABAB receptors.

gamma-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.

P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents.

The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP >> GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones.

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5'-triphosphate (UTP) or adenosine 5'-triphosphate (ATP) inhibits the N-type voltage-gated calcium current by approximately 65%, with an IC50 of 0.5 microM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

Activation of nucleotide receptors inhibits high-threshold calcium currents in NG108-15 neuronal hybrid cells.

UNLABELLED: A P2U (UTP-sensitive) nucleotide receptor has previously been cloned from NG108-15 neuroblastoma x glioma hybrid cells and it has been shown that activation of this receptor inhibits the M-type K+-current. We now report that UTP also inhibits Ca2+-currents in differentiated NG108-15 cells, but probably through a different nucleotide receptor. UTP (100 microM) inhibited the peak of the high-threshold current by 28.4 +/- 3.1% (n = 28) with no effect on the low-threshold current. Two components of high-threshold current were identified: one inhibited by 100 nM omega-conotoxin (CgTx) and one inhibited by 2 microM nifedipine and enhanced by 1 microM BAY K8644. UTP inhibited the former by 31.0 +/- 3.1%, with an IC50 of 2. 8 +/- 1.1 microM, and the latter 34.2 +/- 6.1% with an IC50 of 1.7 +/- 1.3 microM. Pertussis toxin pretreatment prevented inhibition of the CgTx-sensitive, nifedipine-resistant but not CgTx-resistant current. Inhibition was not prevented by intracellular BAPTA (20 mM) or cAMP (1mM). Effects of UTP on both currents were imitated by UDP, ATP, ADP, AP4A and ATPgammaS but weakly or not at all by 2-MeSATP, GTP, AMP-CPP or ITP. Since the receptors which inhibit Ca2+-currents are activated by ATP, it is suggested that they might mediate auto-inhibition of transmitter release by ATP if present on purinergic nerve terminals. KEYWORDS: nucleotides, UTP, ATP, calcium currents, neuroblastoma cells

M-type K+ current inhibition by a toxin fron the scorpion Buthus eupeus.

A number of invertebrate venoms have been tested for effects on M-type K+ currents (IK(M)) in differentiated mouse neuroblastoma X rat glioma NG108-15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited IK(M) by approximately 44% at 50 microgram/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrimidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited IK(M) by approximately 45% with an IC50 of approximately 33 nM. Neither the crude venom nor the purified polypeptide affected the Ca2+ current or the delayed rectifier K+ current. While the crude venom prolonged the Na+ current, the polypeptide did not. Thus, the 4 kDa Buthus eupeus polypeptide appears to be a selective inhibitor of IK(M) in NG108-15 cells.

Activation of nucleotide receptors inhibits M-type K current [IK(M)] in neuroblastoma x glioma hybrid cells.

A phospholipase-C-linked nucleotide receptor, sensitive to both uridine and adenosine triphosphate (UTP and ATP) has been cloned from NG108-15 neuroblastoma x glioma hybrid cells. We have tested whether activation of this receptor could inhibit the voltage-dependent K+ current [IK(M) or "M-current"] in NG108-15 cells recorded using whole-cell patch-clamp methods. Both UTP and ATP inhibited IK(M) by 44% and 42%, respectively, at 100 microM. Mean IC50 values were: UTP, 0.77 +/- 0.27 microM; ATP, 1.81 +/- 0.82 microM. The order of nucleotide and nucleoside activity at 100 microM was: UTP = ATP > ATP [gamma S] = ITP > 2-MeSATP > ADP = GTP >> AMP-CPP, adenosine, where ATP[gamma S] is adenosine 5'-O-(3-thiotriphosphate), ITP is inosine 5'-triphosphate, 2-MeSATP is 2-methylthio ATP and AMP-CPP is alpha, beta methylene ATP. This rank order accords with their activities at the cloned P2U receptor. Effects were not inhibited by suramin (up to 500 microM) or by pre-incubation for 12 h in 500 ng.ml-1 Pertussis toxin. Inhibition of IK(M) was frequently preceded by a transient outward current, probably a Ca(2+)-activated K+ current, responding to Ca2+ mobilization. No effect on the delayed rectifier K+ current was observed. These observations match those expected from stimulating other phospholipase-C-linked receptors in NG108-15 cells.

[Structure-activity study of the basic toxic component of venom from the ant Ectatomma tuberculatum]. / Strukturno-funktsional'noe issledovanie osnovnogo toksicheskogo komponenta iada murav'ia Estatomma tuberculatum.

A toxic principle of the Ectatomma tuberculatum ant venom called ectatomin was isolated. Ectatomin is a protein with molecular weight 7928 Da. Its complete amino acid sequence and spatial structure in aqueous solution were determined by protein chemistry methods and NMR spectroscopy techniques. Ectatomin contains two highly homologous polypeptide chains linked to each other by a disulfide bond. The chains consist of 37 and 34 amino acid residues with an internal disulfide bridge in each. In aqueous solution the molecule forms a bundle of four amphipathic alpha-helices. This toxin in a concentration of 0.05-0.01 mM forms potential dependent nonselective cation channels both in cell and artificial membranes. The channel is dimeric and the mechanism of its formation can be explained in terms of the spatial structure established.

Mechanism of alpha-latrotoxin action as revealed by patch-clamp experiments on Xenopus oocytes injected with rat brain messenger RNA.

Single-channel currents produced by alpha-latrotoxin from the black widow spider venom were recorded on Xenopus oocytes injected with rat brain messenger RNA fraction of 7-8 kb. Single-channel conductance varied from 3 pS to 200 pS and sublevels of similar conductance were observed at both normal and high external concentration of Ca2+. Currents reversed at 0 mV, and the channels were permeable to Ca2+, Na+ and K+ indicating non-selective cation channel produced by the toxin. Ca2+ stabilized the channel mainly at one conducting sublevel. Studies of channel kinetics indicated that openings co-operated into groups of bursts. Within these groups the histograms of closed and open times showed two exponentials with mean times near 1.5 ms and 20 ms for the closed time histogram and 85 ms and 300 ms for the open time histogram at -40 mV. Open times increased with membrane hyperpolarization while closed times did not. Open probability was near 0.8 and slightly increased with hyperpolarization. Elevation of external Ca2+ or toxin concentration promoted the appearance of groups of burst openings while within these groups, the single-channel conductance, the reversal potential and channel kinetics did not depend on Ca2+ or toxin concentration. On the basis of the experimental results, the kinetic mechanism of toxin action has been proposed. The data strongly suggest that alpha-latrotoxin molecules are cation channels associated into clusters that insert into the membrane after binding to the receptor located at active zones of synaptic transmission. Binding and synchronization of channel openings in a cluster are promoted by Ca2+. Influx of Ca2+ through this near permanently open cation channel seems to induce intensive synaptic vesicle fusion and massive neurotransmitter release.

Expression of receptor for alpha-latrotoxin in Xenopus oocytes after injection of mRNA from rat brain.

alpha-Latrotoxin, the major toxin of black widow spider venom, was suggested to bind to the specific receptor on the membrane of presynaptic cells and to activate a nonselective cation channel. The aim of this investigation was to express the receptor to alpha-latrotoxin in the membrane of Xenopus laevis oocytes. Responses to alpha-latrotoxin were studied using a double microelectrode voltage-clamp technique on X. laevis oocytes previously injected with poly(A+)-RNA from rat brain. alpha-Latrotoxin (10 nM) was shown to induce a slow activating reversible inward membrane current at a clamp potential of -60 mV. A second application of alpha-latrotoxin immediately after washing out induced a smaller response. Reversal potential of this current was near to 0 mV; it hardly changed in low Cl- external solution. Response to alpha-latrotoxin did not depend significantly on the variation of Ca2+ concentration in external solution. Ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) injection into oocytes did not decrease alpha-latrotoxin-induced current, but seemed to slow the kinetics of the response. Inorganic Ca-channel blocker Co2+ had no effect on alpha-latrotoxin response. These results indicate that alpha-latrotoxin-induced inward current flows mainly through cation nonspecific channel. A lectin concanavalin A irreversibly inhibited alpha-latrotoxin-evoked inward current. Many of these observations are similar to those reported for nerve cells after alpha-latrotoxin application. The data obtained suggest that functional receptor to alpha-latrotoxin can be successively expressed in the membrane of Xenopus oocytes providing future search of DNA encoding receptor subunits and study of receptor structure-function relationship.

[Potential- and stimulus-dependence of the effect of dihydropyridine Ca-agonists on calcium channels in heart fibers]. / Potentsial- i stimulzavisimosti éffekta digidropiridinovykh Ca-agonistov na kal'tsievye kanaly v voloknalh serdtsa.

It has been shown on the frog auricle fibres by the method of double sucrose bridge that the dependence of the effect of dihydropyridine Ca agonists BAYK 8644, CGP 28 392 and (-S)202 = 791 on calcium channels on the membrane potential is to a large extent due to the activation and following inactivation of Ca channels "silent" in the control. This effect takes place due to a shift by Ca agonists of the stationary curve of channel activation to hyperpolarization. The absence of stimulus-dependence of the agonist effect and constant time of Ca current reactivation suggest that the agonists bind with resting channels.

[Effects on anthracycline antibiotics on ionic currents and contraction of frog atrial fibrils associated with Na+/Ca2+ exchange]. / Vliianie antratsiklinovykh antibiotikov na ionnye toki i sokrashchenievolokon predserdiia liagushki, sviazannoe s Na+/Ca2+-obmenom.

The action of the anthracycline antibiotics rubomycin and its nitroxyl analogue ehoxyl(ruboxyl) at the concentration 100 mg/l was studied in the experiments, have been carried out with the isolated bundles of frog atrium using simultaneous registration of the ionic currents (or the active potentials) and the contraction. It has been shown, that rubomycin caused an action of potential shortening, membrane depolarization, increasing of the background outward current IKI, slowing of the muscle relaxation and the growth of the ionic contraction, associated with the Na/Ca exchange. Nitroxyl derivative of the rubomycin ruboxyl, exhibited small toxicity, did not change the ionic currents and the parameters of the mechanical activity. It is assumed, that cardiotoxicity of anthracyclines is in the great degree due to their ability to block the elimination of the Ca++ from the cell via the Na/Ca exchange, that results in the disturbance of the Ca-homeostasis of the cardiomyocyte and its calcium overload.

Generation of twitch tension in frog atrial fibers by Na/Ca exchange.

Electrical and mechanical responses of frog atrial trabeculae were studied simultaneously using the double-sucrose gap method. Action potentials and twitch tension could be successively generated in fibers in which the slow inward calcium channel current was not observed. As a rule, this could be obtained in the course of a long experiment (3 to 4 hours). Peak tension was shown to increase monotonically with membrane potential in these preparations. In preparations with the slow inward current the total peak tension could be separated into two components. The first component (tonic) monotonically increased with the membrane potential and was probably related to Na/Ca exchange (Horackova 1984). The potential dependency of the second (phasic) component correlated with that of the slow inward calcium current. Only the tonic but not the phasic component could be observed in preparations without the presence of the slow inward calcium current. The tonic component prevailed when both the slow inward current and phasic tension were greatly reduced by nifedipine. Long experiments, long depolarizing clamp pulses, a metabolic inhibitor 2,4-dinitrophenol, inhibitors of Na/K pump ouabain and AR-L57, toxins promoting intracellular sodium accumulation (aconitine, scorpion toxin) were all shown to increase the tonic tension, but not the slow inward current; they induced a transition from biphasic tension-voltage curve into a monotonically increasing one. We concluded that these procedures and agents greatly stimulate Ca influx via Na/Ca exchange. These results show that Na/Ca exchange can function as a reserve system of Ca2+ used for contraction, thus supporting the heart function, especially under unfavourable metabolic conditions.

[Effect of the neurotoxin apamin on ion currents in membranes of heart fibers]. / Vliianie neirotoksina apamina na ionnye toki membran serdechnykh volokon.

It has been shown on auricle fibres of the frog that neurotoxin apamin in extremely low concentrations (10(-10)-10(-9) M) suppresses the current entering in the cell through calcium channels of the membrane, but it increases tonic contraction related to Na/Ca exchange. No significant effects on fast sodium and potassium channels were observed. The results show that the mechanism of apamin effect on the heart is to a large extent conditioned by specific influence on Ca transport through the cell membranes.

[Effect of melittin on ion currents in heart cell membranes]. / Deistvie melittina na ionnye toki membran kletok serdtsa.

It has been shown on auricle fibres of the frog that neurotoxin from bee poison melittin suppresses the ionic currents entering the cell through calcium and sodium channels of the membrane, increases the background potassium current, suppresses phasic and tonic contraction of the fibres. Toxin modifies the kinetics of calcium channels, but does not affect activation and desensitization of beta adrenoreceptors. Effects of melittin are not decreased when adding the inhibitor of phospholipase A2 indomethacin. The results show that melittin directly affects the protein components of the membrane-ionic channels, probably binding with them.