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

Quantitative RT-PCR results show that N-terminal splice isoforms (LCavβ_A and LCavβ_B), but not HOOK domain splice isoforms (LCavβ− and LCavβ+) of snail Cavβ subunits have tissue specific mRNA expression patterns.

<p>mRNA levels are illustrated as fold change relative to HPRT mRNA levels. (A) More generalized pattern of splicing of LCavβ_A containing exons 1a/1b, than LCavβ_B containing exon 2. Exon 2 containing isoform is mostly expressed in the brain, and residual levels in the heart. (B) Exon 7 splicing generates seven extra amino acids (exon 7a− vs exon 7a+) and appears to have no tissue selectivity pattern of expression. (B, inset) Percent of LCavβ− vs LCavβ+, illustrating that 74%–84% of all transcripts lack the extra amino acids in exon 7. (C) LCa_v1 L-type channel has a more generalized mRNA expression pattern as LCavβ_A while more nervous system specific LCa_v2 has a more discrete expression pattern as LCavβ_B isoforms. (D) Rises and falls in the relative fold changes in mRNA levels from juvenile to adult animals are correlated between LCavβ, LCa_v1 and LCa_v2 channel subunits.</p

Conserved exon-intron organization, alternative splicing, and N-terminal intron sizes in the genomic sequence spanning calcium channel beta (Ca_vβ) subunits.

<p>(<b>A</b>) Alignment of the 15 exons of Ca_vβ subunits comparing snail and human Ca_vβ subunit splicing. Ca_vβ subunits have mutually exclusive splicing of N-terminal exon 1a/1b or exon 2 isoforms. Exon 7 in the HOOK domain is subject to mutually-exclusive exon splicing (exon 7a or exon 7b or exon 7c) in vertebrates or splicing in mollusks generated by alternative acceptor sites (exon 7a+, exon 7a−). Molluscan and vertebrate have truncated forms of Ca_vβ subunits lacking the GK domain and C-terminus as a result of skipping of exon 7. (<b>B</b>) Most of the intron sizes of Ca_vβ subunits span the N-terminal exons, and the size of the total intron sequence in the N-terminus increases with the number of exons in the N-termini.</p

Snail Ca_v2 channels nor snail LCa_vβ subunits do not promote the closed-state inactivation observed for mammalian Ca_v2.2 and Ca_vβ₃ or Ca_vβ_1b subunits.

<p>The size of test barium currents relative to the prepulse current after time delays of 0.5, 4, 8, 12, 16, 20 and 40_vβ₃, Ca_vβ_1b, Ca_vβ_2a and snail LCa_vβ_A+ with mammalian Ca_v2.2 or snail LCa_v2 calcium channels. A closed-state inactivation exhibited where there is an increasing inactivation with increasing time delay, is found only with particular combination of subunits, which includes mammalian Ca_v2.2 and Ca_vβ₃ or Ca_vβ_1b<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092941#pone.0092941-Senatore2" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092941#pone.0092941-Senatore3" target="_blank">[19]</a>.</p

Summary of electrophysiology parameters for Figures 5–8.

<p>Summary of electrophysiology parameters for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092941#pone-0092941-g005" target="_blank">Figures 5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092941#pone-0092941-g008" target="_blank">8</a>.</p

Multiple sequence alignments illustrate conserved splicing in (A) the N-terminus and (B) HOOK domain of invertebrates and vertebrate Ca_vβ subunits.

<p>Alternate N-terminal isoforms composed of exons 1a–1b (Ca_vβ_A) or exon 2 (Ca_vβ_B), and HOOK domain splicing includes optional short addendum to exon 7 (Ca_vβ−/Ca_vβ+) in invertebrates or mutually exclusive splicing, exon 7a, 7b, 7c. Note that Ca_vβ from snails, squid, schistosomes and bees and vertebrate Ca_vβ_2a have slow inactivation kinetics and (<b>B</b>) possess HOOK domains with a long form of exon 7 (A form) with a common polybasic region at its 3′ end.</p