Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions.

Invertebrate LCaV3 shares the quintessential features of vertebrate CaV3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca2+ channels, the CaV1 and CaV2 channels. Unlike the vertebrates though, CaV3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCaV3 channel into a higher Na+ and lower Ca2+ current passing channel, more resembling a classical NaV1 Na+ channel. Cnidarian CaV3 T-type channels can possess genes with alternative cysteine-rich, D4L6 extracellular loops in a manner reminiscent of the alternative cysteine-rich, D2L5 extracellular loops of non-cnidarian invertebrates. We illustrate here that the preferences for greater Na+ or Ca2+ ion current passing through CaV3 T-type channels are contributed by paired cysteines within D2L5 and D4L6 extracellular loops looming above the pore selectivity filter. Swapping of invertebrate tri- and tetra-cysteine containing extracellular loops, generates higher Na+ current passing channels in human CaV3.2 channels, while corresponding mono- and di-cysteine loop pairs in human CaV3.2 generates greater Ca2+ current passing, invertebrate LCaV3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCaV3 channels increases relative monovalent ion current sizes and increases the potency of Zn2+ and Ni2+ block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of CaV3.2 channels, including the loss of the slowing of inactivation kinetics during Zn2+ block. Charge neutralization of a ubiquitous aspartate residue of calcium passing CaV1, CaV2 and CaV3 channels, in the outer pore of the selectivity filter residues in Domain II generates higher Na+ current passing channels in a manner that may resemble how the unique D2L5 extracellular loops of invertebrate CaV3 channels may confer a relatively higher peak current size for Na+ ions over Ca2+ The extracellular loops of CaV3 channels are not engaged with accessory subunit binding, as the other Na+ (NaV1) and Ca2+ (CaV1/CaV2) channels, enabling diversity and expansion of cysteine-bonded extracellular loops, which appears to serve, amongst other possibilities, to alter to the preferences for passage of Ca2+ or Na+ ions through invertebrate CaV3 channels.

An ancestral MAGUK protein supports the modulation of mammalian voltage-gated Ca2+ channels through a conserved CaVß-like interface.

Eukaryote voltage-gated Ca2+ channels of the CaV2 channel family are hetero-oligomers formed by the pore-forming CaVα1 protein assembled with auxiliary CaVα2δ and CaVß subunits. CaVß subunits are formed by a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain connected through a HOOK domain. The GK domain binds a conserved cytoplasmic region of the pore-forming CaVα1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between CaV channel subunits in distant eukaryotic organisms by investigating the function of a MAGUK protein (XM_004990081) cloned from the choanoflagellate Salpingoeca rosetta (Sro). This MAGUK protein (Sroß) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian CaVß. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel CaV2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like CaVß, Sroß prevents fast degradation of total CaV2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroß and the AID motif of the pore-forming CaVα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of CaV2.3 with Sroß and functional upregulation of CaV2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroß, similar to the locus found in mammalian CaVß, significantly reduced peak current density. Altogether our data demonstrate that an ancestor MAGUK protein reconstitutes the biophysical and molecular features responsible for channel upregulation by mammalian CaVß through a minimally conserved molecular interface.

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.