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Calcium Channel-Coupled Transcription Factors Facilitate Direct Calcium Signaling
Voltage gated calcium channels (VGCCs) are known to play crucial roles in maintaining neuronal excitability, activity-dependent changes in gene expression, and long-term learning and memory changes within the central nervous system. The main pore-forming alpha subunits (α1 subunits) of these calcium channels are encoded by 10 distinct genes corresponding to the 10 members of this family (Catterall, 2010). Our lab has previously shown that the VGCC gene CACNA1A is a bicistronic cellular gene, producing two functionally distinct proteins from a single mRNA. CACNA1A produces the P/Q-type CaV2.1 VGCC via canonical, cap-dependent translation, and the transcription factor α1ACT through a cryptic Internal ribosome entry site (IRES) within the primary open reading frame (Du et al., 2013; Du et al., 2019). To determine if the L-type VGCC gene CACNA1C and the T-type VGCC gene CACNA1H, representing the two additional major subtypes of VGCC, are bicistronic cellular genes similar to CACNA1A, I employed a series of molecular biology experiments to manipulate the coding sequences of each VGCC gene. Using mRNA transfections, I was able to convincingly demonstrate that both CACNA1C and CACNA1H produce secondary proteins independently of their full-length VGCC α1 subunits via a cap-independent, IRES-like mechanism. Additionally, using molecular manipulations to the CACNA1C and CACNA1H mRNAs, I excluded the possibility that these secondary proteins, termed α1CCT and α1HCT respectively, are produced via other methods of alternative transcription and translation, including alternative splicing of the mRNA, an underlying cryptic promoter in the DNA sequence, or ribosomal skipping/read-through/shunting.
As our lab previously showed that α1ACT functions as a transcription factor, I performed next-generation sequencing studies to investigate the gene regulation capabilities of α1CCT and α1HCT. RNA-seq and ChIP-seq revealed complex networks of genes regulated by each VGCC C-terminal protein (CTP) that promoted neuronal differentiation and synaptic function programs in human neural progenitor cells. Finally, as these novel secondary CTPs are embedded within parent calcium channel genes, I hypothesized that their subcellular localization and possible transcriptional regulation capabilities were regulated in part by neuronal activity. To test this, I employed both neuronal imaging techniques in tandem with depolarizing stimuli to induce calcium influx. In living neurons, calcium spikes induced via glutamate uncaging caused α1CCT and α1ACT to translocate to and from the nucleus, respectively, indicating that these transcription factors are indeed activity dependent. Additionally, pharmacological manipulations showed that calcium influx through specific channels, namely L-type VGCCs and NMDA channels, regulated the intracellular translocation of α1CCT and α1ACT, respectively. These results reveal a conserved method of coordinated protein expression within the VGCC family, as parent VGCC channels are transcriptionally and functionally coupled with their secondary C-terminal transcription factors.
Mutations in these VGCC genes lead to debilitating neurological, muscle, sensory, and cardiac disorders. Loss- or gain-of-function mutations in the CACNA1C gene, encoding the α1C calcium channel subunit of the CaV1.2 L-type VGCC, lead to several pleiotropic disorders including the autism spectrum disorder (ASD) Timothy’s Syndrome, bipolar disorder, schizophrenia, and major depressive disorder (MDD), as well as severe cardiac arrhythmias (Splawski et al., 2004; Green et al., 2009; Dedic et al., 2017; Boczek et al., 2015). Loss- or gain-of-function mutations in the α1H subunit, comprising the CaV3.2 T-type VGCC, are associated with chronic neuropathic pain disorders, ASDs, several types of mental illness, and amyotrophic lateral sclerosis (ALS) (Carter et al., 2019; Becker et al., 2017; Rzhepetskyy et al., 2016;Splawski et al., 2006; Souza et al., 2015). Attempts to recapitulate such phenotypically complex disorders in models has often fallen short, in large part due to the genetic complexity of these disorders and the failure of single-mutation models to capture that complexity. While these results push forward our understanding of the complex pathological mechanisms underlying VGC-mediated disorders, future experiments identifying necessary secondary signaling molecules, as well as the underlying activity-dependent changes to gene expression induced by α1CCT and α1ACT, will lead to a better understanding of this complex transcriptional network in function and dysfunction