Functional Characterization of RNA Editing and Alternative Splicing in the Carboxyl-Terminus of Cav 1.3 Calcium Channel

Abstract

Cav1.3 is a member of the L-type family of voltage-gated calcium channels (LTCC) and is predominantly expressed in the brain, cochlear hair cells, sinoatrial node (SAN), and pancreatic ß-islets. Low-voltage activation of Cav1.3 channels controls excitability in sensory cells and central neurons, as well as pace-making in the SAN. Intramolecular protein interactions in the carboxyl-terminus of Cav1.3 proteins modulate calmodulin binding, altering calcium-dependent inactivation (CDI). Post-transcriptional modification of pre-mRNA, which includes alternative splicing and RNA editing, is vital for the correct translation of the genome and customization of proteins for optimal performance in individual cells. The IQ motif of Cav1.3 channel is edited by Adenosine Deaminases Acting on RNA (ADAR), changing adenosine to inosine at three loci and DNA sequencing analysis showed that guanosine is observed only in the cDNA of Cav1.3. DNA sequencing analysis of cDNA from ADAR2-/- knockout mouse demonstrated that ADAR2 is crucial for RNA editing of Cav1.3. Protein analysis of the Cav1.3 proteins showed that the edited peptides are expressed in the wild-type mouse brain. Immunocytochemistry analysis demonstrated similar surface localization profiles between the edited and wild-type Cav1.3 proteins in primary hippocampal neurons. In addition, RNA editing of the IQ motif in Cav1.3 is central nervous system (CNS)-specific and developmentally regulated. To identify the mechanisms responsible for the CNS-specificity and developmental regulation, neuronal and insulinoma cell lines were examined and found to express only unedited Cav1.3 channels. Experimental manipulations of culture conditions demonstrated that glucose metabolism, neuronal differentiation, availability of cofactor zinc, and transient ADAR2 overexpression were insufficient for promoting editing in Cav1.3, despite elevated ADAR2 activity and its nuclear localization. Full-length analysis of ADAR2 showed higher percent of splice isoform with exon 5a, associated with higher ADAR2 catalytic activity, in the rat brain. Co-expression studies of synthetic construct gIQECS and ADAR2 showed significant editing at two adenosine loci, demonstrating that secondary pre-mRNA structure of Cav1.3 is critical for site-selective editing and cis-acting elements in the cell lines or outside CNS could prevent ADAR2-mediated editing. Using transcript-scanning method, we identified eight different splice variants in the C-terminus of Cav1.3 expressed in rat brain. Electrophysiological characterization of the splice variants demonstrated modulations to activation, inactivation, and recovery properties. A novel C-terminal modulator (CTM) in Cav1.3 is responsible for diminished CDI in the long variant Cav1.342, and a key residual change in the distal C-terminus of rat and human Cav1.3 is critical for this reduction. Correction of this cloning error in our rat clone was sufficient for recapitulating the reported biophysical properties. Skipping of exon 41 removed the IQ motif, abolished CDI completely and decreased current density significantly. Removal of 91 nucleotides in Cav1.343i caused a frame-shift and CTM-deletion, resulting in robust CDI of similar intensity as the short variant Cav1.342a, hyperpolarized shift in activation, and faster recovery from inactivation. Skipping of exon 44 and use of alternative acceptor site at exon 48 resulted in two splice variants that retained both CTM and type I PDZ-binding motif ITTL. However, shortening of the C-termini dampened CDI, caused hyperpolarized shifts in activation, and increased recovery from inactivation. Finally, removal of ITTL motif in exon 42a, ¿41 and exon 43i splice variants did not affect its soma-dendritic localization or synaptic targeting.