The latrophilin family: multiply spliced G protein-coupled receptors with differential tissue distribution.
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Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. We now report the finding of two novel latrophilin homologues. All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini. Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha-latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain-specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions. (+info)
N-type voltage-dependent calcium channels mediate the nicotinic enhancement of GABA release in chick brain.
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The role of voltage-dependent calcium channels (VDCCs) in the nicotinic acetylcholine receptor (nAChR)-mediated enhancement of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) was investigated in chick brain slices. Whole cell recordings of neurons in the lateral spiriform (SpL) and ventral lateral geniculate (LGNv) nuclei showed that cadmium chloride (CdCl2) blocked the carbachol-induced increase of spontaneous GABAergic IPSCs, indicating that VDCCs might be involved. To conclusively show a role for VDCCs, the presynaptic effect of carbachol on SpL and LGNv neurons was examined in the presence of selective blockers of VDCC subtypes. omega-Conotoxin GVIA, a selective antagonist of N-type channels, significantly reduced the nAChR-mediated enhancement of gamma-aminobutyric acid (GABA) release in the SpL by 78% compared with control responses. Nifedipine, an L-type channel blocker, and omega-Agatoxin-TK, a P/Q-type channel blocker, did not inhibit the enhancement of GABAergic IPSCs. In the LGNv, omega-Conotoxin GVIA also significantly reduced the nAChR-mediated enhancement of GABA release by 71% from control values. Although omega-Agatoxin-TK did not block the nicotinic enhancement, L-type channel blockers showed complex effects on the nAChR-mediated enhancement. These results indicate that the nAChR-mediated enhancement of spontaneous GABAergic IPSCs requires activation of N-type channels in both the SpL and LGNv. (+info)
Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis.
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1. In the present study, two new peptides, phrixotoxins PaTx1 and PaTx2 (29-31 amino acids), which potently block A-type potassium currents, have been purified from the venom of the tarantula Phrixotrichus auratus. 2. Phrixotoxins specifically block Kv4.3 and Kv4.2 currents that underlie I(to1), with an 5 < IC50 < 70 nM, by altering the gating properties of these channels. 3. Neither are the Shaker (Kv1), Shab (Kv2) and Shaw (Kv3) subfamilies of currents, nor HERG, KvLQT1/IsK, inhibited by phrixotoxins which appear specific of the Shal (Kv4) subfamily of currents and also block I(to1) in isolated murine cardiomyocytes. 4. In order to evaluate the physiological consequences of the Ito1 inhibition, mice were injected intravenously with PaTx1, which resulted in numerous transient cardiac adverse reactions including the occurrence of premature ventricular beats, ventricular tachycardia and different degrees of atrioventricular block. 5. The analysis of the mouse electrocardiogram showed a dose-dependent prolongation of the QT interval, chosen as a surrogate marker for their ventricular repolarization, from 249 +/- 11 to 265 +/- 8 ms (P < 0.05). 6. It was concluded that phrixotoxins, are new and specific blockers of Kv4.3 and Kv4.2 potassium currents, and hence of I(to1) that will enable further studies of Kv4.2 and Kv4.3 channel and/or I(to1) expression. (+info)
Selective effects of neuronal-synaptobrevin mutations on transmitter release evoked by sustained versus transient Ca2+ increases and by cAMP.
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Synaptobrevin is a key constituent of the synaptic vesicle membrane. The neuronal-synaptobrevin (n-syb) gene in Drosophila is essential for nerve-evoked synaptic currents, but miniature excitatory synaptic currents (mESCs) remain even in the complete absence of this gene. To further characterize the defect in these mutants, we have examined conditions that stimulate secretion. Despite the inability of an action potential to trigger fusion, high K+ saline could increase the frequency of mESCs 4- to 17-fold in a Ca2+-dependent manner, and the rate of fusion approached 25% of that seen in wild-type synapses under the same conditions. Similarly, the mESC frequency in n-syb null mutants could be increased by a Ca2+ ionophore, A23187, and by black widow spider venom. Thus, the ability of the vesicles to fuse in response to sustained increases in cytosolic Ca2+ persisted in the absence of this protein. Tetanic stimulation could also increase the frequency of mESCs, particularly toward the end of a train and after the train of stimuli. In contrast, these mutants did not respond to an elevation of cAMP induced by an activator of adenylyl cyclase, forskolin, or a membrane-permeable analog of cAMP, dibutyryl cAMP, which in wild-type synapses causes a marked increase in the mESC frequency even in the absence of external Ca2+. These results are discussed in the context of models that invoke a special role for n-syb in coupling fusion to the transient, local changes in Ca2+ and an as yet unidentified target of cAMP. (+info)
Novel gating mechanism of polyamine block in the strong inward rectifier K channel Kir2.1.
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Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure. (+info)
Neurexins are functional alpha-latrotoxin receptors.
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Alpha-latrotoxin is a potent neurotoxin that triggers synaptic exocytosis. Surprisingly, two distinct neuronal receptors for alpha-latrotoxin have been described: CIRL/latrophilin 1 (CL1) and neurexin-1alpha. Alpha-latrotoxin is thought to trigger exocytosis by binding to CL1, while the role of neurexin 1alpha is uncertain. Using PC12 cells, we now demonstrate that neurexins indeed function as alpha-latrotoxin receptors that are at least as potent as CL1. Both alpha- and beta-neurexins represent autonomous alpha-latrotoxin receptors that are regulated by alternative splicing. Similar to CL1, truncated neurexins without intracellular sequences are fully active; therefore, neurexins and CL1 recruit alpha-latrotoxin but are not themselves involved in exocytosis. Thus, alpha-latrotoxin is unique among neurotoxins, because it utilizes two unrelated receptors, probably to amplify recruitment of alpha-latrotoxin to active sites. (+info)
Calcium channels involved in synaptic transmission from reticulospinal axons in lamprey.
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The pharmacology of calcium channels involved in glutamatergic synaptic transmission from reticulospinal axons in the lamprey spinal cord was analyzed with specific agonists and antagonists of different high-voltage activated calcium channels. The N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTx) induced a large decrease of the amplitude of reticulospinal-evoked excitatory postsynaptic potentials (EPSPs). The P/Q-type calcium channel blocker omega-agatoxin IVA (omega-Aga) also reduced the amplitude of the reticulospinal EPSPs, but to a lesser extent than omega-CgTx. The dihydropyridine agonist Bay K and antagonist nimodipine had no effect on the amplitude of the reticulospinal EPSP. Combined application of omega-CgTx and omega-Aga strongly decreased the amplitude the EPSPs but was never able to completely block them, indicating that calcium channels insensitive to these toxins (R-type) are also involved in synaptic transmission from reticulospinal axons. We have previously shown that the group III metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) mediates presynaptic inhibition at the reticulospinal synapse. To test if this presynaptic effect is mediated through inhibition of calcium influx, the effect of L-AP4 on reticulospinal transmission was tested before and after blockade of N-type channels, which contribute predominantly to transmitter release at this synapse. Blocking the N-type channels with omega-CgTx did not prevent inhibition of reticulospinal synaptic transmission by L-AP4. In addition, L-AP4 had no affect on the calcium current recorded in the somata of reticulospinal neurons or on the calcium component of action potentials in reticulospinal axons. These results show that synaptic transmission from reticulospinal axons in the lamprey is mediated by calcium influx through N-, P/Q- and R-type channels, with N-type channels playing the major role. Furthermore, presynaptic inhibition of reticulospinal transmission by L-AP4 appears not to be mediated through inhibition of presynaptic calcium channels. (+info)
Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C.
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alpha-latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G-protein-coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin-G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with G alpha q/11 and G alpha o but not with G alpha s, G alpha i or G alpha z, indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX-evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit the Ca(2+)-dependent component of the toxin-evoked release. Based on (i) the known involvement of G alpha q in the regulation of inositol-1,4,5-triphosphate generation and (ii) the requirement for Ca2+ in LTX action, we tested the effect of inhibitors of Ca2+ mobilization on the toxin-evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca(2+)-dependent toxin's action. Thapsigargin, which depletes intracellular Ca2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca2+. On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metabolism do not inhibit the Ca2(+)-independent component of LTX-stimulated release. In the absence of Ca2+, the toxin induces in the presynaptic membrane non-selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca2+ provided intracellular Ca2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca2+, which then triggers secretion. (+info)