FUNCTIONAL MODULATION OF THE CAV3.1 CALCIUM CHANNEL BY ALTERNATIVE SPLICING AT THE C-TERMINUS

The CACNA1G gene encodes for Cav3.1, the alpha-1G subunit of a low-voltage-activated T-type calcium channel, which plays crucial roles in cardiac and smooth muscle cells and neurons by influencing the transmembrane potentials and regulating intracellular Ca2+ signaling. Heterozygous missense mutations in the CACNA1G gene cause spinocerebellar ataxia-42 (SCA42), a heterogeneous group of neurodegenerative disorders characterized by brainstem and cerebellum degeneration, and mice that are null for this gene present severe motor coordination defects, abnormal electrical conductance in the heart, aberrant action potentials in the brain, disrupted sleeping patterns, and absence epilepsy. Different splice variants of Cav3.1 have been described, and alternative splicing of Cav3.1 channel transcripts can alter channel kinetics, localization, and cytosolic Ca2+ trafficking, which create a complex and diverse system of electrical conduction and signal transduction. In this work, we analyze the function of two exons, termed E34 and E35, which are found immediately after domain IV in the intracellular C-terminus, and their function is still largely unknown. We have discovered that these exons are alternatively spliced in all possible combinations in mouse tissues, giving rise to four different splice variants. E34 and E35 are preferentially included in nerve tissue postnatally, while they are mostly skipped in embryonic tissues. To examine the physiological properties of these splice variants, we recorded channel activity with two-electrode voltage clamp on Xenopus oocytes injected with mRNAs corresponding to specific splice variants. Our data show that the Cav3.1 variants that include either or both E34 or E35 produce much larger channel currents than the variant skipping both exons, suggesting that the protein sequences encoded by E34 and E35 may facilitate channel trafficking, or increase the channel open probability or signal channel conductance. To investigate the mechanism of E34 and E35 splicing regulation, we generated minigene reporters and tested them against specific splicing factors that may modulate their inclusion and/or skipping. Our data show that E34 and E35 splicing is regulated by Nova1, Nova2, and Ptbp2, which promote exon inclusion in a dose-dependent manner. We also investigated the effects of the calcium-binding protein calmodulin (CaM) on the channel activity of the different splice variants, and we believe that the C-terminus of Cav3.1 mediates in part the interaction with CaM in mammalian cells transfected with full-length Cav3.1. Moreover, CaM confers current facilitation on exon 34 and/or exon 35-including Cav3.1 variants and a positive shift in the inactivation curves of exon 35-including Cav3.1 variants. Taken together, our data indicate that the alternative splicing at E34 and E35 of Cav3.1 may regulate neuron excitability and modulate the intrinsic firing pattern by regulating the Ca2+ influx through the channel