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The Shaker family of voltage-gated K + channels has been thought of as an animal-specific ion channel family that diversified in concert with nervous systems. It comprises four functionally independent gene subfamilies (Kv1-4) that encode diverse neuronal K + currents. Comparison of animal genomes predicts that only the Kv1 subfamily was present in the animal common ancestor. Here, we show that some choanoflagellates, the closest protozoan sister lineage to animals, also have Shaker family K + channels. Choanoflagellate Shaker family channels are surprisingly most closely related to the animal Kv2-4 subfamilies which were believed to have evolved only after the divergence of ctenophores and sponges from cnidarians and bilaterians. Structural modeling predicts that the choanoflagellate channels share a T1 Zn 2+ binding site with Kv2-4 channels that is absent in Kv1 channels. We functionally expressed three Shakers from Salpingoeca helianthica (SheliKvT1.1-3) in Xenopus oocytes. SheliKvT1.1-3 function only in two heteromultimeric combinations (SheliKvT1.1/1.2 and SheliKvT1.1/1.3) and encode fast N-type inactivating K + channels with distinct voltage dependence that are most similar to the widespread animal Kv1-encoded A-type Shakers. Structural modeling of the T1 assembly domain supports a preference for heteromeric assembly in a 2:2 stoichiometry. These results push the origin of the Shaker family back into a common ancestor of metazoans and choanoflagellates. They also suggest that the animal common ancestor had at least two distinct molecular lineages of Shaker channels, a Kv1 subfamily lineage predicted from comparison of animal genomes and a Kv2-4 lineage predicted from comparison of animals and choanoflagellates., Competing Interests: Competing interests statement:The authors declare no competing interest.

One 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.

Cav3 T-type calcium channels from great pond snail Lymnaea stagnalis have a selectivity-filter ring of five acidic residues, EE(D)DD. Splice variants with exons 12b or 12a spanning the extracellular loop between the outer helix IIS5 and membrane-descending pore helix IIP1 (IIS5-P1) in Domain II of the pore module possess calcium selectivity or dominant sodium permeability, respectively. Here, we use AlphaFold2 neural network software to predict that a lysine residue in exon 12a is salt-bridged to the aspartate residue immediately C terminal to the second-domain glutamate in the selectivity filter. Exon 12b has a similar folding but with an alanine residue in place of lysine in exon 12a. We express LCav3 channels with mutated exons Ala-12b-Lys and Lys-12a-Ala and demonstrate that they switch the ion preference to high sodium permeability and calcium selectivity, respectively. We propose that in the calcium-selective variants, a calcium ion chelated between Domain II selectivity-filter glutamate and aspartate is knocked-out by the incoming calcium ion in the process of calcium permeation, whereas sodium ions are repelled. The aspartate is neutralized by the lysine residue in the sodium-permeant variants, allowing for sodium permeation through the selectivity-filter ring of four negatively charged residues akin to the prokaryotic sodium channels with four glutamates in the selectivity filter. The evolutionary adaptation in invertebrate LCav3 channels highlight the involvement of a key, ubiquitous aspartate, "a calcium beacon" of sorts in the outer pore of Domain II, as determinative for the calcium ion preference over sodium ions through eukaryotic Cav1, Cav2, and Cav3 channels., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A 400-level undergraduate oral presentation and discussion course in Systems Neuroscience was delivered asynchronously online during the COVID-19 pandemic. Enrolled students banked their narrated oral presentations in video format online then engaged in peer evaluation in virtual classrooms through the course website. Student delivered their oral presentation and responded to peer questions at their leisure and convenience, without the stress and anxiety associated with a "live" performance delivery in front of their peers. A remote and asynchronously delivered course facilitated much more peer contact than "live" versions of the course, which included a total of 62 uploaded presentations, 301 video responses uploaded to 1985 questions posed by peers, a total of 1159 feedback questionnaires submitted, 1066 rankings submitted of viewed oral presentations, and 1091 scores submitted evaluating the quality of questions posed by reviewers of oral presentations. A major drawback in the remote, asynchronous deliver was the enormity of peer engagement through the course website portal, which was mostly blind to the instructor because of the inability to effectively cross-index data linked between the student entries in the LEARN course website and the uploaded videos stored within BONGO Video Assignment tool. Nonetheless, a consistent engagement of students, and the positive feedback from enrolled students, indicate that a future version of this oral/written discussion course will be delivered, in part, remotely and asynchronously, even without a mandated delivery of the course by a remote and asynchronous method due to the COVID-19 pandemic restrictions in 2020-2021.

Eukaryote voltage-gated Ca 2+ channels of the Ca V 2 channel family are hetero-oligomers formed by the pore-forming Ca V α1 protein assembled with auxiliary Ca V α2δ and Ca V β subunits. Ca V β 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 Ca V α1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between Ca V 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 (Sroβ) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian Ca V β. Recombinant expression of its cDNA with mammalian high-voltage activated Ca 2+ channel Ca V 2.3 in mammalian HEK cells produced robust voltage-gated inward Ca 2+ currents with typical activation and inactivation properties. Like Ca V β, Sroβ prevents fast degradation of total Ca V 2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroβ and the AID motif of the pore-forming Ca V α1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of Ca V 2.3 with Sroβ and functional upregulation of Ca V 2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroβ, similar to the locus found in mammalian Ca V β, 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 Ca V β through a minimally conserved molecular interface., Competing Interests: Declaration of competing interest The authors declare that they do not have any conflict of interest with the contents of this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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 (Na v 1 and Na v 2) and calcium channels (Ca v 1, Ca v 2, and Ca v 3) 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 Na v 1 channels to adapt to sodium ion selectivity and fast ion gating. A non-selective, and slowly gating Na v 2 channel homolog in single cell eukaryotes, predate the diversification of Na v 1 channels from a basal homolog in a common ancestor to extant cnidarians to the nine vertebrate Na v 1.x channel genes plus Nax. A close kinship between Na v 2 and Na v 1 homologs is evident in the sharing of most (twenty) intron splice junctions. Different metazoan groups have lost their Na v 1 channel genes altogether, while vertebrates rapidly expanded their gene numbers. The expansion in vertebrate Na v 1 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 Na v 1 channels within different animal sub-phyla. Na v 1 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.

Calcium (Ca v 1 and Ca v 2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C termini, which associate with calmodulin (CaM), a universal calcium sensor. Ca v 3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCa v 3) and human Ca v 3.1, Ca v 3.2, and Ca v 3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I-II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high-nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM in 1 H- 15 N HSQC NMR spectra. Intact Ca 2+ -binding sites on CaM and an intact gating brake sequence (first 39 amino acids of the I-II linker) were required in Ca v 3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Ca v 3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4-12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel.

The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE) and calcium (EEEE) selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM) was lowered to 0.1 mM and were inhibited (>40% to >90%) with 10 µM Gd3+, 100 µM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (<30%) to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 µM mibefradil or 100 µM nifedipine. We hypothesize that the rapid changes in leak current size in response to changing external cations or drugs relates to their influences on the membrane seal adherence and the electro-osmotic flow of mobile cations channeling in crevices of a particular pore size in the interface between the negatively charged patch electrode and the lipid membrane. Observed sodium leak conductance currents in weak patch seals are reproducible between the electrode glass interface with cell membranes, artificial lipid or Sylgard rubber.

T-type (Cav3) channels are categorized as calcium channels, but invertebrate ones can be highly sodium-selective channels. We illustrate that the snail LCav3 T-type channel becomes highly sodium-permeable through exon splicing of an extracellular turret and descending helix in domain II of the four-domain Cav3 channel. Highly sodium-permeable T-type channels are generated without altering the invariant ring of charged residues in the selectivity filter that governs calcium selectivity in calcium channels. The highly sodium-permeant T-type channel expresses in the brain and is the only splice isoform expressed in the snail heart. This unique splicing of turret residues offers T-type channels a capacity to serve as a pacemaking sodium current in the primitive heart and brain in lieu of Nav1-type sodium channels and to substitute for voltage-gated sodium channels lacking in many invertebrates. T-type channels would also contribute substantially to sodium leak conductances at rest in invertebrates because of their large window currents.

The accessory beta subunit (Ca(v)β) 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)β subunits (β1, β2, β3, β4) 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)β subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCa(v)1, LCa(v)2, and LCa(v)β). Invertebrate Ca(v)β 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 β2a 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)β subunit. LCa(v)β 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)β subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa(v)β 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 Ca(v)2.2 and Ca(v)β3 subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCa(v)2 nor LCa(v)β) appears to be compatible with this observed property.

Cav3 T-type channels are low-voltage-gated channels with rapid kinetics that are classified among the calcium-selective Cav1 and Cav2 type channels. Here, we outline the fundamental and unique regulators of T-type channels. An ubiquitous and proximally located "gating brake" works in concert with the voltage-sensor domain and S6 alpha-helical segment from domain II to set the canonical low-threshold and transient gating features of T-type channels. Gene splicing of optional exon 25c (and/or exon 26) in the short III-IV linker provides a developmental switch between modes of activity, such as activating in response to membrane depolarization, to channels requiring hyperpolarization input before being available to activate. Downstream of the gating brake in the I-II linker is a key region for regulating channel expression where alternative splicing patterns correlate with functional diversity of spike patterns, pacemaking rate (especially in the heart), stage of development, and animal size. A small but persistent window conductance depolarizes cells and boosts excitability at rest. T-type channels possess an ion selectivity that can resemble not only the calcium ion exclusive Cav1 and Cav2 channels but also the sodium ion selectivity of Nav1 sodium channels too. Alternative splicing in the extracellular turret of domain II generates highly sodium-permeable channels, which contribute to low-threshold sodium spikes. Cav3 channels are more ubiquitous among multicellular animals and more widespread in tissues than the more brain centric Nav1 sodium channels in invertebrates. Highly sodium-permeant Cav3 channels can functionally replace Nav1 channels in species where they are lacking, such as in Caenorhabditis elegans.

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 Ca(2+) 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 Ca(2+)-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.

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN's most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd ( 3+) -sensitive, NMDG (+) -impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (ILP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Nax or Cav 1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels.

NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE) or sodium channels (EKEE or EEKE). NALCN channels with alternative calcium, (EEEE) and sodium, (EKEE or EEKE) -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K)-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG(+) impermeant and blockable with 10 µM Gd(3+) ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2):371-83.

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, LCa(v)3 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 Ca(v)3 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 Ca(v)3.1 and encompassing the proximal half of the I-II linker, imparts a ~50% reduction in total and surface-expressed LCa(v)3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Ca(v)3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development.

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&#95;3o18, F&#95;3i10, and I&#95;4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q&#95;3o18, Y&#95;3i10, and M&#95;4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N&#95;3o18&#95;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.

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28°C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28°C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37°C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28°C for further incubation. We find that the initial incubation at 28°C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

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.

Here we describe features of the first non-mammalian T-type calcium channel (LCa(v)3) expressed in vitro. This molluscan channel possesses combined biophysical properties that are reminiscent of all mammalian T-type channels. It exhibits T-type features such as "transient" kinetics, but the "tiny" label, usually associated with Ba(2+) conductance, is hard to reconcile with the "bigness" of this channel in many respects. LCa(v)3 is 25% larger than any voltage-gated ion channel expressed to date. It codes for a massive, 322-kDa protein that conducts large macroscopic currents in vitro. LCa(v)3 is also the most abundant Ca(2+) channel transcript in the snail nervous system. A window current at typical resting potentials appears to be at least as large as that reported for mammalian channels. This distant gene provides a unique perspective to analyze the structural, functional, drug binding, and evolutionary aspects of T-type channels.

Rem2 belongs to the RGK family of small GTPases whose members are known to interact with the voltage gated calcium channel beta subunit, and to inhibit or abolish calcium currents. To identify the underlying functional domains of Rem2, we created several N- or C-terminally truncated Rem2 proteins and examined their abilities to interact with the Ca(v) beta subunit and to regulate the activities of Ca(v)2.2 N-type calcium channels. Confocal imaging of Rem2 in tsA-201 cells revealed that it contains a membrane-targeting signal in its C-terminus, consistent with previous studies. Co-precipitation assays showed that Ca(v) beta(3) interaction depends on Rem2 residues 1-123. Only Rem2 proteins that targeted the cell membrane as well as bound the beta subunit were able to reduce whole cell calcium currents.

Activation of protein kinase C (PKC) potentiates secretion in Aplysia peptidergic neurons, in part by inducing new sites for peptide release at growth cone terminals. The mechanisms by which ion channels are trafficked to such sites are, however, not well understood. We now show that PKC activation rapidly recruits new Ca(V)2 subunits to the plasma membrane, and that recruitment is blocked by latrunculin B, an inhibitor of actin polymerization. In contrast, inhibition of microtubule polymerization selectively prevents the appearance of Ca(V)2 subunits only at the distal edge of the growth cone. In resting neurons, Ca(V)2-containing organelles reside in the central region of growth cones, but are absent from distal lamellipodia. After activation of PKC, these organelles are transported on microtubules to the lamellipodium. The ability to traffic to the most distal sites of channel insertion inside the lamellipodium does, therefore, not require intact actin but requires intact microtubules. Only after activation of PKC do Ca(V)2 channels associate with actin and undergo insertion into the plasma membrane.

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the alpha-type, 1 is of the beta-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The alpha-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise approximately 10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.

L-type calcium channel activity has been associated with a number of cytoplasmic responses, including gene transcription and activation of calcium-dependent enzymes, yet their direct contribution to the electrical activities of neurons has remained largely unexplored. Here we report the cloning and functional characterization of a molluscan L-type calcium channel homologue, LCa(v)1, and investigate its role in coordinating neuronal firing patterns. The LCav1 channel exhibits many hallmarks of vertebrate L-type channels in that it is high-voltage activated, slowly inactivating, and dihydropyridine sensitive and displays calcium-dependent inactivation in recording solutions with standard EGTA concentrations. We show that despite comprising less than approximately 20% of the total whole cell current in identified Lymnaea respiratory network neurons, the L-type channels are essential for maintaining rhythmic action potential discharges without being involved in synaptic release. Our data therefore suggest an important role of L-type calcium channels in maintaining rhythmical pattern activity underlying breathing behavior in Lymnaea.

Calcium channel beta subunits are key modulators of calcium channel function and membrane targeting of the pore-forming alpha1 subunit. Here we show that an invertebrate (Lymnaea stagnalis) homolog of P/Q- and N-type calcium channels (LCav2), although colocalized with beta subunits in synapses of mature neurons, is physically uncoupled from the beta subunits in the leading edge of growth cones of outgrowing neurons. Moreover, LCav2 channels that mediate transmitter release in mature synapses also participate in neuronal outgrowth in growth cones. The differential association of beta subunits with synaptic calcium channels and those expressed in emergent neuronal growth suggests that beta subunits may play a role in the transformation of Cav2 calcium channel function in immature neurons and mature synapses., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca2+-triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca2+ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca2+ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.

Here we report the first assessment of the expression and modulation of an invertebrate alpha1 subunit homolog of mammalian presynaptic Cav2 calcium channels (N-type and P/Q-type) in mammalian cells. Our data show that molluscan channel (LCav2a) isolated from Lymnaea stagnalis is effectively membrane-targeted and electrophysiologically recordable in tsA-201 cells only when the first 44 amino acids of LCav2a are substituted for the corresponding region of rat Cav2.1. When coexpressed with rat accessory subunits, the biophysical properties of LCav2a-5'rbA resemble those of mammalian N-type calcium channels with respect to activation and inactivation, lack of pronounced calcium dependent inactivation, preferential permeation of barium ions, and cadmium block. Consistent with reports of native Lymnaea calcium currents, the LCav2a-5'rbA channel is insensitive to micromolar concentrations of omega-conotoxin GVIA and is not affected by nifedipine, thus confirming that it is not of the L-type. Interestingly, the LCav2a-5'rbA channel is almost completely and irreversibly inhibited by guanosine 5'-3-O-(thio)triphosphate but not regulated by syntaxin1, suggesting that invertebrate presynaptic calcium channels are differently modulated from their vertebrate counterparts.

In vertebrates, the physical coupling between presynaptic calcium channels and synaptic vesicle release proteins enhances the efficiency of neurotransmission. Recent evidence indicates that these synaptic proteins may feedback directly on synaptic release by negatively regulating calcium entry, and indirectly through pathways involving second messenger molecules. Studies of individual neurons from both vertebrates and invertebrates have provided novel insights into the roles of scaffolding proteins in calcium channel targeting and neurotransmitter release. These studies require us to expand current models of synaptic transmission.

The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (approximately 3 microm), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaea requires the expression of a C-terminal splice variant of the Lymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.

The amplitude of an A-like potassium current (I(Kfast)) in identified cultured motor neurons isolated from the jellyfish Polyorchis penicillatus was found to be strongly modulated by extracellular potassium ([K(+)](out)). When expressed in Xenopus oocytes, two jellyfish Shaker-like genes, jShak1 and jShak2, coding for potassium channels, exhibited similar modulation by [K(+)](out) over a range of concentrations from 0 to 100 mM. jShak2-encoded channels also showed a decreased rate of inactivation and an increased rate of recovery from inactivation at high [K(+)](out). Using site-directed mutagenesis we show that inactivation of jShak2 can be ascribed to an unusual combination of a weak "implicit" N-type inactivation mechanism and a strong, fast, potassium-sensitive C-type mechanism. Interaction between the two forms of inactivation is responsible for the potassium dependence of cumulative inactivation. Inactivation of jShak1 was determined primarily by a strong "ball and chain" mechanism similar to fruit fly Shaker channels. Experiments using fast perfusion of outside-out patches with jShak2 channels were used to establish that the effects of [K(+)](out) on the peak current amplitude and inactivation were due to processes occurring at either different sites located at the external channel mouth with different retention times for potassium ions, or at the same site(s) where retention time is determined by state-dependent conformations of the channel protein. The possible physiological implications of potassium sensitivity of high-threshold potassium A-like currents is discussed.

The jellyfish gene, jShak2, coded for a potassium channel that showed increased conductance and a decreased inactivation rate as [K(+)](out) was increased. The relative modulatory effectiveness of K(+), Rb(+), Cs(+), and Na(+) indicated that a weak-field-strength site is present. Cysteine substituted mutants (L369C and F370C) of an N-terminal truncated construct, (jShak2Delta2-38) which only showed C-type inactivation, were used to establish the position and nature of this site(s). In comparison with jShak2Delta2-38 and F370C, L369C showed a greater relative increase in peak current when [K(+)](out) was increased from 1 to 100 mM because the affinity of this site was reduced at low [K(+)](out). Increasing [K(+)](out) had little effect on the rate of inactivation of L369C; however, the appearance of a second, hyperbolic component to the inactivation curve for F370C indicated that this mutation had increased the affinity of the low-affinity site by bringing the backbone oxygens closer together. Methanethiosulphonate reagents were used to form positively (MTSET), negatively (MTSES), and neutrally (MTSM) charged side groups on the cysteine-substituted residues at the purported K(+) binding site(s) in the channel mouth and conductance and inactivation kinetic measurements made. The reduced affinity of the site produced by the mutation L369C was probably due to the increased hydrophobicity of cysteine, which changed the relative positions of carbonyl oxygens since MTSES modification did not form a high-field-strength site as might be expected if the cysteine residues project into the pore. Addition of the side chain -CH(2)-S-S-CH(3), which is similar to the side chain of methionine, a conserved residue in many potassium channels, resulted in an increased peak current and reduced inactivation rate, hence a higher affinity binding site. Modification of cysteine substituted mutants occurred more readily from the inactivated state confirming that side chains probably rotate into the pore from a buried position when no K ions are in the pore. In conclusion we were able to show that, as for certain potassium channels in higher taxonomic groups, the site(s) responsible for modulation by [K(+)](out) is situated just outside the selectivity filter and is represented by the residues L(369) and F(370) in the jellyfish Shaker channel, jShak2.

1. When jellyfish Shaker potassium channels (jShak2) are heterologously expressed in Xenopus oocytes at different levels they demonstrate density-dependent changes in electrical and kinetic properties of macroscopic currents. 2. The activation and inactivation properties of jShak2 channels depend on the extracellular potassium concentration. In this study we present experimental data which show that expression-dependent changes in kinetic and electrical properties of jShak2 macroscopic currents can be explained by the positive feedback effect of dynamic accumulation of K+ in the perimembranal space.

Voltage-gated Na+ channels are responsible for fast propagating action potentials. The structurally simplest animals known to contain rapid, transient, voltage-gated currents carried exclusively by Na+ ions are the Cnidaria. The Cnidaria are thought to be close to the origin of the metazoan radiation and thus are pivotal organisms for studying the evolution of the Na+ channel gene. Here we describe the genomic organization of the Na+ channel alpha subunit, PpSCN1, from the hydrozoan jellyfish, Polyorchis penicillatus. We show that most of the 20 intron sites in this diploblast are conserved in mammalian Na+ channel genes, with some even shared by Ca2+ channels. One of these conserved introns is spliced by a rare U 12-type spliceosome. Such conservation places the origin of the primary exon arrangement of Na+ channels and different intron splicing mechanisms to at least the common ancestors of diploblasts and triploblasts, approximately 600 million-1 billion years ago.

Extant cnidarians are probably the simplest metazoans with discrete nervous systems and rapid, transient voltage-gated currents carried exclusively by Na+ ions. Thus cnidarians are pivotal organisms for studying the evolution of voltage-gated Na+ channels. We have isolated a full-length Na+ channel alpha subunit cDNA (PpSCN1) from the hydrozoan jellyfish, Polyorchis penicillatus, that has one of the smallest known coding regions of a four domain Na+ channel (1695 amino acids). Homologous residues that have a critical bearing on the selectivity filter, voltage-sensor and binding sites for tetrodotoxin and lidocaine in vertebrates and most invertebrates differ in cnidarians. PpSCN1 is not alternatively-spliced and may be the only pore-forming alpha subunit available to account for at least three electrophysiologically distinct Na+ currents that have been studied in P. penicillatus.

The S4 segment of the jellyfish (Polyorchis penicillatus) Shaker channel jShak1 contains only six positively charged motifs. All other Shaker channels, including the jellyfish Shaker channel jShak2, have seven charges in this segment. Despite their charge differences, both these jellyfish channels produce currents with activation and inactivation curves shifted by approximately +40 mV relative to other Shaker currents. Adding charge without changing segment length by mutating the N-terminal side of jShak1 S4 does not have a pronounced effect on channel activation properties. Adding the positively charged motif RIF on the N-terminal side of K294 (the homologue of K374 in Drosophila Shaker, which is a structurally critical residue) produced a large positive shift in both activation and inactivation without altering the slope of the activation curve of the channel. When IFR was added to the other side of K294, there was a small negative shift in activation and fast inactivation of the channel was prevented. Our results demonstrate that K294 divides the S4 segment into functionally different regions and that the voltage threshold for activation and inactivation of the channel is not determined by the total charge on S4.

1. Whole cell voltage-clamp recordings from isolated swimming motor neurons (SMNs) reveal a rapidly activating and inactivating sodium current. 2. Permeability ratios of PLi/PNa = 0.941 and P(guanidinium)/PNa = 0.124 were measured for the mediating channel, which was impermeable to rubidium. 3. The conductance/voltage and steady state inactivation curves are shifted in a depolarizing direction by approximately 45 mV relative to most neuronal sodium currents in higher animals. 4. Activation could be fitted with two exponents and maximal current peaked at 0.74 +/- 0.06 ms (mean +/- SD). 5. Inactivation could be fitted with fast (Tau 1 = 1.91 +/- 0.07 ms at +10 mV) and slow (Tau 2 = 11.65 +/- 0.55 ms at +10 mV) exponents. 6. Half-recovery from inactivation occurred slowly (52.6 +/- 2.9 ms). 7. A second class of identifiable neurons, "B" neurons, possesses a distinctly different population of sodium channels. they showed different inactivation kinetics and far more rapid recovery from inactivation (half-recovery < 5 ms). 8. We conclude that there was physiological diversification of sodium channels early in metazoan evolution and that there has been considerable cell-specific selection of channel properties.