Retinoic acid affects calcium signaling in adult molluscan neurons.

Retinoic acid, the active metabolite of vitamin A, is important for nervous system development, regeneration, as well as cognitive functions of the adult central nervous system. These central nervous system functions are all highly dependent on neuronal activity. Retinoic acid has previously been shown to induce changes in the firing properties and action potential waveforms of adult molluscan neurons in a dose- and isomer-dependent manner. In this study, we aimed to determine the cellular pathways by which retinoic acid might exert such effects, by testing the involvement of pathways previously shown to be affected by retinoic acid. We demonstrated that the ability of all-trans retinoic acid (atRA) to induce electrophysiological changes in cultured molluscan neurons was not prevented by inhibitors of protein synthesis, protein kinase A or phospholipase C. However, we showed that atRA was capable of rapidly reducing intracellular calcium levels in the same dose- and isomer-dependent manner as shown previously for changes in neuronal firing. Moreover, we also demonstrated that the transmembrane ion flux through voltage-gated calcium channels was rapidly modulated by retinoic acid. In particular, the peak current density was reduced and the inactivation rate was increased in the presence of atRA, over a similar time course as the changes in cell firing and reductions in intracellular calcium. These studies provide further evidence for the ability of atRA to induce rapid effects in mature neurons.

Gene splicing of an invertebrate beta subunit (LCavß) in the N-terminal and HOOK domains and its regulation of LCav1 and LCav2 calcium channels.

The accessory beta subunit (Ca(v)ß) of calcium channels first appear in the same genome as Ca(v)1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Ca(v)ß subunits (ß1, ß2, ß3, ß4) which associate with four Ca(v)1 channel isoforms (Ca(v)1.1 to Ca(v)1.4) and three Ca(v)2 channel isoforms (Ca(v)2.1 to Ca(v)2.3). Here we assess the fundamentally-shared features of the Ca(v)ß subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCa(v)1, LCa(v)2, and LCa(v)ß). Invertebrate Ca(v)ß subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Ca(v)2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate ß2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCa(v)ß subunit. LCa(v)ß will also slow the inactivation kinetics of LCa(v)3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Ca(v)ß subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa(v)ß subunits have an N-terminal "A" isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate ß1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable "B" N-terminus (exon 2) in the exon position of mammalian ß3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Ca(v)2.2 and Ca(v)ß3 subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCa(v)2 nor LCa(v)ß) appears to be compatible with this observed property.

Mapping of dihydropyridine binding residues in a less sensitive invertebrate L-type calcium channel (LCa v 1).

Invertebrate L-type calcium channel, LCa(v) 1, isolated from the pond snail Lymnaea stagnalis is nearly indistinguishable from mammalian Ca(v) 1.2 (α1C) calcium channel in biophysical characteristics observed in vitro. These L-type channels are likely constrained within a narrow range of biophysical parameters to perform similar functions in the snail and mammalian cardiovascular systems. What distinguishes snail and mammalian L-type channels is a difference in dihydropyridine sensitivity: 100 nM isradipine exhibits a significant block of mammalian Ca(v) 1.2 currents without effect on snail LCa(v)1 currents. The native snail channel serves as a valuable surrogate for validating key residue differences identified from previous experimental and molecular modeling work. As predicted, three residue changes in LCa(v)1 (N_3o18, F_3i10, and I_4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q_3o18, Y_3i10, and M_4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N_3o18_Q mutation in LCa(v) 1 channels lowers DHP sensitivity even further and the triple mutation bearing enhanced isradipine sensitivity, still retains a reduced potency of agonist, (S)-Bay K8644.

G-proteins modulate invertebrate synaptic calcium channel (LCav2) differently from the classical voltage-dependent regulation of mammalian Cav2.1 and Cav2.2 channels.

Voltage-gated calcium channels in the Ca(v)2 channel class are regulators of synaptic transmission and are highly modified by transmitter inputs that activate synaptic G-protein-coupled receptors (GPCRs). A ubiquitous form of G-protein modulation involves an inhibition of mammalian Ca(v)2.1 and Ca(v)2.2 channels by Gbetagamma dimers that can be relieved by high-frequency trains of action potentials. Here, we address whether the ubiquitous and versatile form of G-protein regulation in mammals is also found in simpler invertebrate nervous systems. Remarkably, the invertebrate LCa(v)2 channel from the pond snail, Lymnaea stagnalis, does not bear any of the hallmarks of mammalian, voltage-dependent G-protein inhibition of Ca(v)2.2. Swapping either the I-II linker or N-terminus of Ca(v)2.2, which serve as key binding domains for G-protein inhibition, does not endow invertebrate LCa(v)2 channels with voltage-dependent G-protein modulatory capacity. Instead, in vitro expressed LCa(v)2 channels are inhibited slowly by the activation of cAMP, in a manner that depends on G-proteins but does not depend on Gbetagamma subunits. A similar G-protein and cAMP-dependent inhibition of nifedipine-insensitive LCa(v)2 currents is also consistent in native and identified Lymnaea VD4 neurons. The slower inhibition using a cellular messenger such as cAMP may meet the modulatory needs in invertebrates while an activity-dependent regulation, evolving in vertebrates, provides a more dynamic, fine-tuning of neurosecretion by regulating the influence of neurotransmitter inputs through presynaptic GPCRs.