P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.

Abstract

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the a-conotoxin-and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channd (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N-or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K+-induced Ca'5 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FIX and sFVX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.Ca2' influx through voltage-dependent Ca2' channels (VDCCs) is the trigger for the release of neurotransmitters from the nerve terminals (1, 2). Three major types ofVDCC named T, L, and N were described in neuronal cells (3). The high-threshold L and N VDCCs are sensitive to the blocking effect of o-conotoxin (c-CgTX), and only the L type is affected by Ca2`channel antagonists of the 1,4-dihydropyridine (DHP) class. An intermediate-threshold VDCC channel called the P channel was identified in the; Purkinje cells of mammalian cerebellum and found to be insensitive to DHP and w-CgTX, but very sensitive to a low molecular weight fraction of the venom of the funnel-web spider Agelenopsis aperta (4). This funnel-web spider toxin (FTX) was also effective in blocking Ca2+ conductance and synaptic transmission at the squid giant synapse (4). Evoked release of neurotransmitter was shown to be dependent on Ca2+ influx through the N-type VDCC in sympathetic neurons by the inhibitory effect of w-CgTX and the lack of effect of DHP (5). By contrast, substance P release from dorsal root ganglia neurons (6, 7) and catecholamine release from chromaffin cells (8) are strongly inhibited by DHP, consistent with a major participation of L-type channels. However, mammalian motor nerve terminals are normally insensitive to either w-CgTX or DHP (9-11). Furthermore, in brain synaptosomes, K+-evoked Ca2' uptake and transmitter release are only partially sensitive to c-CgTX and DHP (12, 13). Thus the identity of the VDCC involved in transmitter release in the majority of the synapses at the mammalian central and peripheral nervous system has not been defined. The experiments presented here were designed to study the effect of FTX on transmitter release and Ca2+ influx at the mammalian neuromuscular junction and on Ca2+ uptake by cerebral cortex synaptosomes in order to determine whether a particular type of VDCC ...

Down‐regulation of non‐L‐, non‐N‐type (Q‐like) Ca2+ channels by Lambert‐Eaton myasthenic syndrome (LEMS) antibodies in rat insulinoma RINm5F cells

“…The presence of a significant fraction of non-L, non-N-type Ca2+ channels in RINm5F cells, with biophysical and pharmacological properties close to the Q-type channel, makes this cell line a suitable model to investigate the inhibitory action of LEMS IgGs on the P/Q-type channels controlling the presynaptic Ca*+ entry in mammalian neuromuscular junctions [9]. ACh release in motor nerve terminals is insensitive to N-and L-type channel blockers [6,7] while it is reversibly abolished by nanomolar concentrations of o-Aga-IVA and by lo*-fold higher concentrations of w-CTx-MVIIC [S].…”

“…Fax: (39) (11) 6707708. E-mail: carbone@unito.it agatoxin IVA [8] and funnel-web spider toxin [9], i.e. by a P/Q-type channel [lo].…”

Autoimmune disorders of the neuromuscular junction

“…ω-Conotoxin (ω-CmTx) is a snail toxin that binds to the P/Q-type VGCC and inhibits neuromuscular transmission. [72] Antibodies in LEMS can be detected by radioimmunoprecipitation of VGCCs extracted from human or mammalian cerebellum that are labeled with 125 1-ω-Conotoxin (ω-CmTx) MVIIC. [73,74] The antibodies are present in over 85% of patients and are highly specific for the disease, although they can be found in some patients with SCLC without LEMS.…”

Mechanisms of Exocytosis in Insulin‐Secreting B‐Cells and Glucagon‐Secreting A‐Cells

“…Despite many similarities of the exocytotic machinery in (neuro-) endocrine cells and that in neurotransmitter-releasing fast synapses, there are important differences: 1) synaptic vesicles are small (diameter Ͻ30 nm; von Gersdorff 1996) compared with peptide containing vesicles (hundreds of nm; Plattner et al 1997;Olofsson et al 2002); 2) synaptic exocytosis occurs without delay after the influx of Ca 2π and is at least one order-of magnitude faster than peptide secretion (Ämmälä et al 1993;Mennerick & Matthews 1996); 3) synaptic vesicles may stay largely intact during exocytosis and are refilled with new transmitter molecules immediately after endocytosis, whereas peptide-containing granules are assembled and filled with secretory peptides in the Golgi apparatus (Betz & Bewick 1992;Hutton 1994); 4) synaptic exocytosis generally requires higher concentrations of Ca 2π (Ämmälä et al 1993;Mennerick & Matthews 1996, Chow et al 1996Bollmann et al 2000); and 5) although most secretory cells contain more than one type of voltage-gated Ca 2π -channel, synaptic transmitter exocytosis depends on the faster N-or P-type (Hirning et al 1988;Uchitel et al 1992), whereas endocrine cells preferentially use the L-type (Lopez et al 1994;Barg et al 2001a). Taken together, the findings in neurones suggest that the site of Ca 2π -influx is within minimal distance of the Ca 2π -sensor at the synaptic vesicle and functionally associated within release sites.…”