Developmental and cell-specific expression ofCacna1dsplice variants

Abstract

CaV1.3 is an L-type voltage-gated calcium channel implicated in several functions including gene expression, pacemaking activity, and neurotransmitter release. The gene that encodes the CaVa1-pore forming subunit of CaV1.3 (Cacna1d) is a multi-exon gene that undergoes extensive alternative splicing, which provides functional versatility to this gene across tissues and cell-types. The function and expression of several Cacna1d splice variants within the C-terminus have been previously characterized. These splice variants differ in their voltage-dependence of activation, Ca 2+ -dependent inactivation, and their sensitivity to dihydropyridines. However, less is known about alternatively spliced exons in Cacna1d located downstream of domain I and upstream of the C-terminus (e11, e22a/e22, e31a/e31b/e32). Here, we performed a systematic study to determine the developmental and cell-specific expression of several Cacna1d splice variants. We found that the cassette e11 is upregulated during brain development, and in adult cortical tissue is more abundant in excitatory neurons relative to inhibitory interneurons. This exon is also upregulated upon nerve growth factor (NGF) induced differentiation of pheochromocytoma cells, PC12. At the functional level, the splice variants resulting from e11 alternative splicing (+e11-Cacna1d and De11-Cacna1d) form functional CaV1.3 channels with similar biophysical properties in expression mammalian systems. Of the pair of mutually exclusive exons, e22a and e22, the later dominates at all stages. However, we observed a slight upregulation of e22 from embryonic to adult human brain. A second pair of mutually exclusive exons, e31a and e31b, was also studied. We found that e31a increases during brain development. Finally, the cassette exon 32 is repressed in adult brain tissue. KEYWORDSCalcium channels, alternative splicing, splicing factors, brain development Parkinson's disease, primary aldosteronism and autism spectrum disorder (Chan et al., 2010;Baig et al., 2011;Kang et al., 2012;Reinbothe et al., 2013; Scholl et al., 2013; Pinggera et al., 2015). Interestingly, Cacna1d splice variants add functional versatility of CaV1.3 channels across tissues and cell-types.CaV1.3, like all CaVa1 subunits, is organized into four homologous domains (DI-IV). Each domain contains six transmembrane-spanning segments (S1-S6), with a reentrant loop between S5 and S6, which contains glutamates that are key for the selectivity filter. The amino and carboxyl termini, as well as linker sequences between the DI-II, DII-III, and DIII-IV are cytosolic (Fig. 1A). Sites of alternative splicing of sequences encoding for the N-terminus, DI, DI-II linker, DIII, DIV and C-terminus are present in Cacna1d pre-mRNAs. Alternative splicing of sequences encoding for the C-terminus in CaV1.3 have been well characterized, and results in splice isoforms with differences in calciumdependent inactivation, trafficking, protein-protein interactions, and sensitivity to dihydropyridines (DHPs) (Shen et al., 2006;Singh et al., 200...

Stabilization of negative activation voltages of Cav1.3 L-Type Ca2+-channels by alternative splicing

“…In a recent unpublished report, LaCarubba and colleagues systematically also analyzed the expression of exons 11 and 32 in mouse whole brains at different postnatal ages as well as in fetal and adult human whole brains. They could show that both are upregulated during development and are therefore dominant variants in adult mouse and human brains [38]. Here we could confirm the expression of exons 11 and 32 not only in the brain but also in MCCs and in the cochlea.…”

“…Previous biophysical characterizations of the role of exons 11 and 32 were not conclusive suggesting no gating changes by both exons when expressed in Xenopus oocytes [21], no changes for exon 11 when expressed in tsA-201 cells [38] (both measured in 5 mM Ba 2+ ) or a modest depolarizing shift in V 0.5,act of +3 mV (i.e. the opposite effect as reported here, measured in 10 mM Ba 2+ ) upon inclusion of exon 32 [22].…”