No abstract

Voltage-dependent calcium channels mediate calcium entry into neurons, which is crucial for many processes in the brain including synaptic transmission, dendritic spiking, gene expression and cell death. Many types of calcium channels exist in mammalian brains, but high-affinity blockers are available for only two types, L-type channels (targeted by nimodipine and other dihydropyridine channel blockers) and N-type channels (targeted by omega-conotoxin). In a search for new channel blockers, we have identified a peptide toxin from funnel web spider venom, omega-Aga-IVA, which is a potent inhibitor of both calcium entry into rat brain synaptosomes and of 'P-type' calcium channels in rat Purkinje neurons. omega-Aga-IVA will facilitate characterization of brain calcium channels resistant to existing channel blockers and may assist in the design of neuroprotective drugs.

No abstract

The Ca 2؉ channel ␣1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca 2؉ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. ␣1A-Subunits are thought to support both P-and Q-type Ca 2؉ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca 2؉ delivery system at excitatory nerve terminals. We generated ␣1A-deficient mice (␣1A ؊͞؊ ) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying Ϸ3-4 weeks after birth. P-type currents in Purkinje neurons and P-and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca 2؉ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in ␣1A ؊͞؊ hippocampal slices persisted despite the lack of P͞Q-type channels but showed enhanced reliance on N-type and R-type Ca 2؉ entry. The ␣1A ؊͞؊ mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in ␣1A. The ␣ 1A -subunit, the most abundant ␣ 1 -subunit in vertebrate brain (1), mediates Ca 2ϩ influx across presynaptic and somatodendritic membranes, thereby triggering fast neurotransmitter release and other key neuronal responses (2-5). Because of its high expression levels in the brain, the ␣ 1A -subunit was the first representative of its subclass to be isolated by cDNA cloning (1, 6). This predominantly neuronal subclass also includes ␣ 1B (N-type Ca 2ϩ channel) and ␣ 1E [possibly R-type Ca 2ϩ channel (7-9)] and is referred to as ABE or Ca V 2. There is no information to date on the behavioral or electrophysiological consequences of deleting a member of the ABE subfamily.␣ 1A Transcripts are widely distributed in rat (10) and human brain (11), most prominently in cell body layers in cerebellum and hippocampus. At the subcellular level, ␣ 1A immunoreactivity has been found in cell bodies, dendrites, and presynaptic terminals (12). Less clear has been the role of ␣ 1A in supporting Ca 2ϩ channel components defined by biophysical and pharmacological criteria. In either Xenopus oocytes (13, 14) or HEK293 cells (15), expression of ␣ 1A -subunits along with ancillary ␣ 2 ͞␦-and ␤-subunits generated currents with properties closely resembling the Q-type current found in cerebellar granule cells (8) and much less the P-type current first described in cerebellar Purkinje neurons by Llinás and colleagues (16,17). Unlike native P-type channels (18), the expressed currents showed pronounced inactivation during sustained depolarizations and responded to -agatoxin IVA (-Aga-IVA) at half-blocking doses of Ϸ100 nM, not Ϸ1 nM (13). Various explanations for the discrepancies have been advanced...

Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.

No abstract

G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous “orphan” GPCRs have become available for functional analysis. Here we characterize two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of −PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were constructed and tested. The insect PRXa peptides are evolutionarily related to the vertebrate peptide neuromedin U (NMU), or are related to arginine vasopressin (AVP), both of which have PRXa motifs. Seven Drosophila GPCRs related to receptors for NMU and AVP were cloned and expressed in Xenopus oocytes for functional analysis. Four Drosophila GPCRs in the NMU group (CG11475, CG8795, CG9918, CG8784) are activated by insect PRXa pyrokinins, (−FXPRXamide), Cap2b-like peptides (−FPRXamide), or ecdysis triggering hormones (−PRXamide). Three Drosophila GPCRs in the vasopressin receptor group respond to crustacean cardioactive peptide (CCAP), corazonin, or adipokinetic hormone (AKH), none of which are PRXa peptides. These findings support a theory of coevolution for NMU and Drosophila PRXa peptides and their respective receptors

To determine whether the different types of Ca2+ channels present in the same secretory cell contribute equally to secretion, we used chromaffin cells to analyse the coupling between three distinct types of Ca2+ channel and exocytosis. These are omega-conotoxin-GVIA-sensitive N-type channels, omega-agatoxin-IVA-sensitive P-type Ca2+ channels and dihydropyridine-sensitive facilitation Ca2+ channels, which are normally quiescent but are activated by depolarizing pre-pulses, repetitive depolarizations to physiological potentials, or agents that raise cyclic AMP. We have simultaneously monitored changes in capacitance as an assay of catecholamine secretion, and Ca2+ currents. Although all three types of Ca2+ channel trigger secretion individually, facilitation channels produce much greater secretion for a given size of Ca2+ current, indicating that they are coupled more efficiently to exocytosis. These results indicate that facilitation Ca2+ channels may be physically nearer vesicle release sites. They also show that low efficiency P- and N-type channels could trigger mild release and that high-efficiency facilitation channels may underlie the massive catecholamine release that occurs during the 'fight or flight' response.