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

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

International audienceThe 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 (\textgreater40% to \textgreater90%) with 10 microM Gd3+, 100 microM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (\textless30%) to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 microM mibefradil or 100 microM 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

Correction: 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. 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

HEK-293T cell currents from a leaky patch (I_LP) have a characteristic drug blocking profile characteristic of ion channel currents.

<p>Bath application of di- and trivalent ions, and calcium channel blockers and their block of I_LP during a 1 second voltage ramp. (A) Representative leaky patch currents. Axes cross at zero. X = voltage (mV), Y = current (pA). (B) Data points (left) and box plot (mean +/− SEM, right).</p

Ionic currents generated through a leaky patch (I_LP) generates a sodium leak conductance which is potentiated in low external calcium and is highly sensitive to Gd³⁺ block.

<p>Left: Architecture of the cell attached patch as detailed by Fredrick Sachs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098808#pone.0098808-Suchyna1" target="_blank">[20]</a>. The membrane seal is the 10–50 micron of adherent membrane scaled along the inside walls of the glass pipette. The negatively charged glass and negatively-charged membrane generates a seal pocket that is filled with a lubricated layer of cations. The electro-osmotic flow of mobile cations in the seal space is the motor driving membrane patch creep. Right: Extracellular treatments that regulate whole cell leak currents in the patch seal. Less mobile cations like NMDG⁺ slows the inward electro-osmotic flow of ions compared to Na⁺ ions. Gd³⁺ has a high local charge density that titrates positive charges into the patch seal space, neutralizing the electrostatic repulsion of the negatively charged glass electrode and membrane, improving the seal resistance, and causing a decrease in leak current size. Lowering Ca²⁺ reduces the adhesive force between the glass and membrane and causes increases in leak current sizes.</p

Percent change in current size of leaky patch current (I_LP) inward current (measured at −100 mV in ramp protocol) and I_LP outward current (measured at +100 mV in ramp protocol) in response to NMDG replacement of external Na ions or 0.1 mM Ca replacement of 1.2 mM external calcium.

<p>Data points (left) and box plot (mean +/− SEM, right). Note that the relative change is remarkably consistent despite the dramatic difference in current densities of I_LP ranging from 0.38 to 171.28 pA/pF, with average 36.70+/−2.93 in 18 HEK-293T cells.</p