Eukaryotic Voltage-Gated Sodium Channels: On Their Origins, Asymmetries, Losses, Diversification and Adaptations

The appearance of voltage-gated, sodium-selective channels with rapid gating kinetics was a limiting factor in the evolution of nervous systems. Two rounds of domain duplications generated a common 24 transmembrane segment (4 × 6 TM) template that is shared amongst voltage-gated sodium (Nav1 and Nav2) and calcium channels (Cav1, Cav2, and Cav3) and leak channel (NALCN) plus homologs from yeast, different single-cell protists (heterokont and unikont) and algae (green and brown). A shared architecture in 4 × 6 TM channels include an asymmetrical arrangement of extended extracellular L5/L6 turrets containing a 4-0-2-2 pattern of cysteines, glycosylated residues, a universally short III-IV cytoplasmic linker and often a recognizable, C-terminal PDZ binding motif. Six intron splice junctions are conserved in the first domain, including a rare U12-type of the minor spliceosome provides support for a shared heritage for sodium and calcium channels, and a separate lineage for NALCN. The asymmetrically arranged pores of 4x6 TM channels allows for a changeable ion selectivity by means of a single lysine residue change in the high field strength site of the ion selectivity filter in Domains II or III. Multicellularity and the appearance of systems was an impetus for Nav1 channels to adapt to sodium ion selectivity and fast ion gating. A non-selective, and slowly gating Nav2 channel homolog in single cell eukaryotes, predate the diversification of Nav1 channels from a basal homolog in a common ancestor to extant cnidarians to the nine vertebrate Nav1.x channel genes plus Nax. A close kinship between Nav2 and Nav1 homologs is evident in the sharing of most (twenty) intron splice junctions. Different metazoan groups have lost their Nav1 channel genes altogether, while vertebrates rapidly expanded their gene numbers. The expansion in vertebrate Nav1 channel genes fills unique functional niches and generates overlapping properties contributing to redundancies. Specific nervous system adaptations include cytoplasmic linkers with phosphorylation sites and tethered elements to protein assemblies in First Initial Segments and nodes of Ranvier. Analogous accessory beta subunit appeared alongside Nav1 channels within different animal sub-phyla. Nav1 channels contribute to pace-making as persistent or resurgent currents, the former which is widespread across animals, while the latter is a likely vertebrate adaptation

Gene Splicing of an Invertebrate Beta Subunit (LCav?) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels

The accessory beta subunit (Cavβ) of calcium channels first appear in the same genome as Cav1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Cavβ subunits (β1, β2, β3, β4) which associate with four Cav1 channel isoforms (Cav1.1 to Cav1.4) and three Cav2 channel isoforms (Cav2.1 to Cav2.3). Here we assess the fundamentally-shared features of the Cavβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCav1, LCav2, and LCavβ). Invertebrate Cavβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Cav2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCavβ subunit. LCavβ will also slow the inactivation kinetics of LCav3 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 Cavβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCavβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a 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 β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Cav2.2 and Cavβ3 subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCav2 nor LCavβ) appears to be compatible with this observed property

Gene Transcription and Splicing of T-Type Channels Are Evolutionarily-Conserved Strategies for Regulating Channel Expression and Gating

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCav3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa) or a short exon 25c (9 aa) in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III–IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Cav3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Cav3.1 and encompassing the proximal half of the I–II linker, imparts a ∼50% reduction in total and surface-expressed LCav3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Cav3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development

Comparison of Promoter Hypermethylation Pattern in Salivary Rinses Collected with and without an Exfoliating Brush from Patients with HNSCC

Background: Salivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. Methodolgy: 57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening an

Docking of LDCVs Is Modulated by Lower Intracellular [Ca2+] than Priming

Many regulatory steps precede final membrane fusion in neuroendocrine cells. Some parts of this preparatory cascade, including fusion and priming, are dependent on the intracellular Ca2+ concentration ([Ca2+]i). However, the functional implications of [Ca2+]i in the regulation of docking remain elusive and controversial due to an inability to determine the modulatory effect of [Ca2+]i. Using a combination of TIRF-microscopy and electrophysiology we followed the movement of large dense core vesicles (LDCVs) close to the plasma membrane, simultaneously measuring membrane capacitance and [Ca2+]i. We found that a free [Ca2+]i of 700 nM maximized the immediately releasable pool and minimized the lateral mobility of vesicles, which is consistent with a maximal increase of the pool size of primed LDCVs. The parameters that reflect docking, i.e. axial mobility and the fraction of LDCVs residing at the plasma membrane for less than 5 seconds, were strongly decreased at a free [Ca2+]i of 500 nM. These results provide the first evidence that docking and priming occur at different free intracellular Ca2+ concentrations, with docking efficiency being the most robust at 500 nM

A governance of ion selectivity based on the occupancy of the “beacon” in one- and four-domain calcium and sodium channels

ABSTRACTOne of nature’s exceptions was discovered when a Cav3 T-type channel was observed to switch phenotype from a calcium channel into a sodium channel by neutralizing an aspartate residue in the high field strength (HFS) +1 position within the ion selectivity filter. The HFS+1 site is dubbed a “beacon” for its location at the entryway just above the constricted, minimum radius of the HFS site’s electronegative ring. A classification is proposed based on the occupancy of the HFS+1 “beacon” which correlates with the calcium- or sodium-selectivity phenotype. If the beacon is a glycine, or neutral, non-glycine residue, then the cation channel is calcium-selective or sodium-permeable, respectively (Class I). Occupancy of a beacon aspartate are calcium-selective channels (Class II) or possessing a strong calcium block (Class III). A residue lacking in position of the sequence alignment for the beacon are sodium channels (Class IV). The extent to which animal channels are sodium-selective is dictated in the occupancy of the HFS site with a lysine residue (Class III/IV). Governance involving the beacon solves the quandary the HFS site as a basis for ion selectivity, where an electronegative ring of glutamates at the HFS site generates a sodium-selective channel in one-domain channels but generates a calcium-selective channel in four-domain channels. Discovery of a splice variant in an exceptional channel revealed nature’s exploits, highlighting the “beacon” as a principal determinant for calcium and sodium selectivity, encompassing known ion channels composed of one and four domains, from bacteria to animals

LCa_v3 channel from snail is a distant homolog to mammalian Ca_v3.1, Ca_v3.2 and Ca_v3.3, with conserved structural features in the cytoplasmic loops.

<p>(A) Phylogeny of T-type calcium channels using maximum parsimony with bootstrap scores indicated on branches. (B) Percent similarity of amino acid sequences of T-type channels (snail vs. human) using the Needleman-Wunsch global alignment algorithm. (C) Running average of similarity of snail versus human T-type channel amino acid sequences using a window of 25 aa (plotcon, EMBOSS <a href="http://emboss.open-bio.org/" target="_blank">http://emboss.open-bio.org/</a>). (D) Alignment of T-type channel sequences in the I–II linker. Amino acids between the Histidine rich region and the APRASPE motif vary from 0 to 89 aa. Accession/IDs: (<i>Trichoplax</i> JGI:21513; <i>Nematostella</i> JGI:170705; <i>Drosophila</i> NM_001103419; <i>Lymnaea</i> AAO83843; <i>Biomphalaria</i> WUGI: Contig251; <i>Nasonia</i> XM_001604352; <i>Bombus</i> XM_003398839; <i>Anolis</i> XM_003217306; <i>Gallus</i> XM_001232653; human Ca_v3.1 NP_061496.2; human Ca_v3.2 NP_066921.2; human Ca_v3.3 NP_066919.2).</p

Summary of biophysical parameters of LCav3 channel variants containing exons 8b and 25c expressed in HEK-293T cells, with one-way analysis of variance to assess statistical significance.

<p>n.s. not significant;</p>*<p>p<0.05;</p>**<p>p<0.005;</p>***<p>p<0.001.</p

Primers used in quantitative RT-PCR experiment (Figure3).

<p>Primers used in quantitative RT-PCR experiment (Figure3).</p