NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

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.

Invertebrate LCa V 3 shares the quintessential features of vertebrate Ca V 3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca 2+ channels, the Ca V 1 and Ca V 2 channels. Unlike the vertebrates though, Ca V 3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCa V 3 channel into a higher Na + and lower Ca 2+ current passing channel, more resembling a classical Na V 1 Na + channel. Cnidarian Ca V 3 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 Ca 2+ ion current passing through Ca V 3 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 Ca V 3.2 channels, while corresponding mono-and di-cysteine loop pairs in human Ca V 3.2 generates greater Ca 2+ current passing, invertebrate LCa V 3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCa V 3 channels increases relative monovalent ion current sizes and increases the potency of Zn 2+ and Ni 2+ block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of Ca V 3.2 channels, including the loss of the slowing of inactivation kinetics during Zn 2+ block. Charge neutralization of a ubiquitous aspartate residue of calcium passing Ca V 1, Ca V 2 and Ca V 3 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 Ca V 3 channels may confer a relatively higher peak current size for Na + ions over Ca 2+ The extracellular loops of Ca V 3 channels are not engaged with accessory subunit binding, as the other Na + (Na V 1) and Ca 2+ (Ca V 1/Ca V 2) 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 Ca 2+ or Na + ions through invertebrate Ca V 3 channels. High field strength site D2L5 Extracellular loop spanning the end of transmembrane segment 5 to the start of the pore selectivity filter (S5-P) in Domain II D4L6 Extracellular loop spanning the end of the pore selectivity filter to the start of transmembrane segment 6 (P-S6

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In recent years, researchers have leveraged the ubiquitin-proteasome system (UPS) to induce selective degradation of proteins by E3 ubiquitin ligases, which has great potential as novel therapeutics for human diseases, including cancer and neurodegenerative disorders. However, despite extensive efforts, only a handful of ~600 human E3 ligases were utilized, and numerous protein–protein interaction surfaces on E3 ligases were not explored. To tackle these problems, we leveraged a structure-based protein engineering technology to develop a multi-domain fusion protein bringing functional E3 ligases to the proximity of a target protein to trigger its proteasomal degradation, which we termed Ubiquitin Variant Induced Proximity (UbVIP). We first generated non-inhibitory synthetic UbV binders for a selected group of human E3 ligases. With these UbVs employed as E3 ligase engagers, we designed a library of UbVIPs targeting a DNA damage response protein 53BP1. We observed that two UbVIPs recruiting RFWD3 and NEDD4L could effectively induce proteasome degradation of 53BP1 in human cell lines. This provides a proof-of-principle that UbVs can act as a means of targeted degradation for nucleus-localized proteins. Our work demonstrated that UbV technology is suitable to develop protein-based molecules for targeted degradation and can help identify novel E3 ligases for future therapeutic development.