A broad survey of choanoflagellates revises the evolutionary history of the Shaker family of voltage-gated K channels in animals.

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.

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

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 "" 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 which correlates with the calcium- or sodium-selectivity phenotype.

Self-Ownership and the Duty to Assist.

Libertarians are attracted to the self-ownership thesis because it seems to satisfy four important theoretical . First, the thesis treats all persons equally by assigning them the same initial set of rights. Second, the thesis gives people the strongest set of ownership rights possible.

A lysine residue from an extracellular turret switches the ion preference in a Cav3 T-Type channel from calcium to sodium ions.

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.

Asynchronous Delivery of a 400 Level, Partially Peer-Graded, Oral Presentation and Discussion Course in Systems Neuroscience for 60 Students during the COVID-19 Pandemic.

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 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.

Investigating the Distribution of Strains of and Streptomycin Resistance in Apple Orchards in New York Using Clustered Regularly Interspaced Short Palindromic Repeat Profiles: A 6-Year Follow-Up.

Fire blight, caused by the bacterium , is one of the most important diseases of apple. The antibiotic streptomycin is routinely used in the commercial apple industries of New York (NY) and New England to manage the disease. In 2002 and again, from 2011 to 2014, outbreaks of streptomycin resistance (SmR) were reported and investigated in NY.

An ancestral MAGUK protein supports the modulation of mammalian voltage-gated Ca channels through a conserved Caβ-like interface.

Eukaryote voltage-gated Ca channels of the Ca2 channel family are hetero-oligomers formed by the pore-forming Caα1 protein assembled with auxiliary Caα2δ and Caβ subunits. Caβ 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 Caα1 subunit referred as the "AID".

Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in Ca3 T-type channels alter the passage and block of monovalent and divalent ions.

Invertebrate LCa3 shares the quintessential features of vertebrate Ca3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca channels, the Ca1 and Ca2 channels. Unlike the vertebrates though, Ca3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCa3 channel into a higher Na and lower Ca current passing channel, more resembling a classical Na1 Na channel. Cnidarian Ca3 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.

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 (Na1 and Na2) and calcium channels (Ca1, Ca2, and Ca3) 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.

Calmodulin regulates Ca3 T-type channels at their gating brake.

Calcium (Ca1 and Ca2) 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. Ca3 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.

Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels.

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.

Gd3+ and calcium sensitive, sodium leak currents are features of weak membrane-glass seals in patch clamp recordings.

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.

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

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.

T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.

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.

Cav3 T-type channels: regulators for gating, membrane expression, and cation selectivity.

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.

The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.

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.

A uniquely adaptable pore is consistent with NALCN being an ion sensor.

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.

NALCN ion channels have alternative selectivity filters resembling calcium channels or sodium 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).

Leidenfrost levitation: beyond droplets.

Friction is a major inhibitor in almost every mechanical system. Enlightened by the Leidenfrost effect - a droplet can be levitated by its own vapor layer on a sufficiently hot surface - we demonstrate for the first time that a small cart can also be levitated by Leidenfrost vapor. The levitated cart can carry certain amount of load and move frictionlessly over the hot surface.

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, 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.

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.

Optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in HEK-293T cells.

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.

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.

Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis.

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.