We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in <i>Lymnaea stagnalis</i> occurs independent of a physical interaction between presynaptic calcium channels and functional complement SNARE proteins. Instead, <i>Lymnaea</i>requires the expression C-terminal splice variant the<i>Lymnaea</i> homolog to mammalian N- P/Q-type channels. show alternately spliced region physically interacts with scaffolding proteins Mint1 CASK, is abolished following...

The Conus magus peptide toxin ω-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and now in clinical trials as intrathecal analgesic. Here, we have examined the action on mutant wild type channels transiently expressed tsA-201 cells. Although shown previously that mutations a putative external EF-hand motif domain IIIS5–H5 region alters block by both GVIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. Zamponi, W. (2001) J. Biol....

NALCN is a member of the family ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges placozoans mostly remains single compared to which diversified into twenty genes in humans. The has alternatively-spliced exons at 15 or exon 31 splices novel selectivity filter residues resemble (EEEE) (EKEE EEKE). alternative calcium, sodium, EEKE)...

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed residues with high helix-forming propensity within mostly disordered N-terminus that conserved in L-type calcium channels from protostome invertebrates to humans. an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes CaM more ancient, C-terminal IQ domain on channels. bound N- C- terminal tails serve as dual detectors changing intracellular Ca2+ concentrations, promoting...

The Shaker family of voltage-gated K

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 that display high sequence similarity to nAChR subunits, which 10 are alpha-type, 1 is beta-type, and was not classified because insufficient information. Heterologous expression confirms their capacity form functional receptors responding acetylcholine. The alpha-type...

The appearance of voltage-gated, sodium-selective channels with rapid gating kinetics was a limiting factor in the evolution nervous systems. Two rounds domain duplications generated common 24 transmembrane segment (4 × 6 TM) template that is shared amongst voltage-gated sodium (Nav1 and Nav2) calcium (Cav1, Cav2, Cav3) leak channel (NALCN) plus homologs from yeast, different single-cell protists (heterokont unikont) algae (green brown). A architecture 4 TM include an asymmetrical...

Here we describe features of the first non-mammalian T-type calcium channel (LCav3) expressed in vitro. This molluscan possesses combined biophysical properties that are reminiscent all mammalian channels. It exhibits such as "transient" kinetics, but "tiny" label, usually associated with Ba2+ conductance, is hard to reconcile "bigness" this many respects. LCav3 25% larger than any voltage-gated ion date. codes for a massive, 322-kDa protein conducts large macroscopic currents also most...

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates vertebrates. The snail gene, LCa(v)3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice large exon 8b (201 aa) or short 25c (9 cytoplasmic linkers, similar to mammalian homologs. Inclusion optional exons III-IV linker speeds up kinetics causes hyperpolarizing shifts both activation...

T-type (Cav3) channels are categorized as calcium channels, but invertebrate ones can be highly sodium-selective channels. We illustrate that the snail LCav3 channel becomes sodium-permeable through exon splicing of an extracellular turret and descending helix in domain II four-domain Cav3 channel. Highly generated without altering invariant ring charged residues selectivity filter governs The sodium-permeant expresses brain is only splice isoform expressed heart. This unique offers a...

Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps exocytosis. Here we use a stage-specific preparation test roles SNAREs, synaptotagmin, and SNARE-binding in mechanism Ca2+-triggered fusion. Excess exogenous proteins, sufficient block SNARE interactions, did not inhibit either Ca2+ sensitivity, extent, kinetics In contrast, despite limited effect on synaptotagmin densities, treatments with high doses chymotrypsin markedly...

L-type calcium channel activity has been associated with a number of cytoplasmic responses, including gene transcription and activation calcium-dependent enzymes, yet their direct contribution to the electrical activities neurons remained largely unexplored. Here we report cloning functional characterization molluscan homologue, LCa(v)1, investigate its role in coordinating neuronal firing patterns. The LCav1 exhibits many hallmarks vertebrate channels that it is high-voltage activated,...

Activation of protein kinase C (PKC) potentiates secretion in Aplysia peptidergic neurons, part by inducing new sites for peptide release at growth cone terminals. The mechanisms which ion channels are trafficked to such are, however, not well understood. We now show that PKC activation rapidly recruits Ca V 2 subunits the plasma membrane, and recruitment is blocked latrunculin B, an inhibitor actin polymerization. In contrast, inhibition microtubule polymerization selectively prevents...

Here we report the first assessment of expression and modulation an invertebrate alpha1 subunit homolog mammalian presynaptic Cav2 calcium channels (N-type P/Q-type) in cells. Our data show that molluscan channel (LCav2a) isolated from Lymnaea stagnalis is effectively membrane-targeted electrophysiologically recordable tsA-201 cells only when 44 amino acids LCav2a are substituted for corresponding region rat Cav2.1. When coexpressed with accessory subunits, biophysical properties...

The properties of leaky patch currents in whole cell recording HEK-293T cells were examined as a means to separate these control from expressed sodium and calcium leak channel snail NALCN channels possessing both (EKEE) (EEEE) selectivity filters. Leak generated by the weakening gigaohm seals artificial membrane rupture using ZAP function on clamp amplifier. Surprisingly, we found that weakened membrane/glass seal can be surprisingly stable exhibit behavior is consistent with current derived...

Abstract T‐type channels are unique among the voltage‐gated calcium in their fast kinetics and low voltages of activation inactivation, latter two features allowing them to operate at near resting membrane potential most neurons. can therefore be recruited by subthreshold depolarizations, hyperpolarizations that remove inactivation. As such, significantly influence how when cells reach action threshold, thus critical regulators excitability. also conserved within animal kingdom, present even...

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture four homologous domains consisting six transmembrane helices, separated by three cytoplasmic linkers and delimited N C-terminal ends. one shortest members, lacking much variation that provides diverse palate gating features, tissue specific adaptations sodium calcium channels. NALCN's most distinctive feature it possesses highly adaptable pore with...

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T must be plated onto glass coverslips at low enough density so that they are not contact with each other order to allow for without confounding effects due adjacent cells. Transfected also express high efficiency the plasma membrane whole-cell patch clamp detectable currents above noise levels....

Calcium channel beta subunits are key modulators of calcium function and membrane targeting the pore-forming alpha1 subunit. Here we show that an invertebrate (Lymnaea stagnalis) homolog P/Q- N-type channels (LCav2), although colocalized with in synapses mature neurons, is physically uncoupled from leading edge growth cones outgrowing neurons. Moreover, LCav2 mediate transmitter release also participate neuronal outgrowth cones. The differential association synaptic those expressed emergent...

Voltage-gated calcium channels in the Ca(v)2 channel class are regulators of synaptic transmission and highly modified by transmitter inputs that activate G-protein-coupled receptors (GPCRs). A ubiquitous form G-protein modulation involves an inhibition mammalian Ca(v)2.1 Ca(v)2.2 Gbetagamma dimers can be relieved high-frequency trains action potentials. Here, we address whether versatile regulation mammals is also found simpler invertebrate nervous systems. Remarkably, LCa(v)2 from pond...