[Immunocytochemical identification of synaptotagmin 1, syntaxin 1, N-type Ca(2+)-channel and nicotinic cholinoreceptor in motor neuromuscular junctions of earthworm somatic muscle Lumbricus terrestris].

The somatic muscle of earthworm contains myoneural synapses forming clusters of "synaptic buttons". In these "buttons", the proteins syntaxin 1, synaptotagmin 1 and alpha 1B subunit of the Ca(2+)-channel of N-type were identified. We suppose that "synaptic buttons" contain a limited number of active zones due to their small size (1-2 microm) and the pattern of distribution of proteins of exo-endocytotic cycle. The postsynaptic membrane of cholinetgic synapses contains nicotinic acetylcholine receptors capable to bind alpha-bungarotoxin. The area of location of receptors on postsynaptic membrane is strictly limited to the region of synaptic contact.

Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis.

Ca(V)2.2 voltage-gated calcium channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with Ca(V)2.2 in purified presynaptic terminal membranes. A number of known and new Ca(V)2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-Ca(V)2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct Ca(V)2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of Ca(V)2.2 was beyond reasonable doubt. The identification of the long-splice Ca(V)2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested.

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling.

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.

Mapping of RGS12-Cav2.2 channel interaction.

The alpha1 (pore-forming) subunit of the Cav2.2 (N-type) channel is tyrosine phosphorylated by Src kinase upon activation of GABAB receptors. The tyrosine-phosphorylated form of the alpha1 subunit of the Cav2.2 channel becomes a target for the binding of RGS12, a GTPase-accelerating protein. Binding of the phosphotyrosine-binding domain of RGS12 to the tyrosine-phosphorylated channel alters the kinetics of the termination of GABA-mediated inhibition of the calcium current. Using a combination of biochemical and electrophysiological approaches, we have determined that the SNARE binding or "synprint" region of the Cav2.2 binds to RGS12. This article describes the protocols used to map the interaction using primary neuronal cultures.