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The syndromic autism spectrum disorder (ASD) Timothy syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Cav1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Cav1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Cav1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Cav1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.

This chapter contains sections titled: Alternative Pre‐mRNA Splicing Alternative Splicing in the Ca v 2 Genes Future Directions Conclusions Acknowledgments References

RNA splicing is a key mechanism linking genetic variation with psychiatric disorders. Splicing profiles are particularly diverse in brain and difficult to accurately identify and quantify. We developed a new approach to address this challenge, combining long-range PCR and nanopore sequencing with a novel bioinformatics pipeline. We identify the full-length coding transcripts of CACNA1C in human brain. CACNA1C is a psychiatric risk gene that encodes the voltage-gated calcium channel CaV1.2. We show that CACNA1C’s transcript profile is substantially more complex than appreciated, identifying 38 novel exons and 241 novel transcripts. Importantly, many of the novel variants are abundant, and predicted to encode channels with altered function. The splicing profile varies between brain regions, especially in cerebellum. We demonstrate that human transcript diversity (and thereby protein isoform diversity) remains under-characterised, and provide a feasible and cost-effective methodology to address this. A detailed understanding of isoform diversity will be essential for the translation of psychiatric genomic findings into pathophysiological insights and novel psychopharmacological targets.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions 1 . TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A 2 – 6 . We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed 7 , we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. Antisense oligonucleotides effectively decrease the inclusion of exon  8A of CACNA1C in human cells both in vitro and in rodents transplanted with human brain organoids, and a single intrathecal administration rescued both calcium changes and in vivo dendrite morphology of patient neurons.

Background The degree to which alternative RNA splicing influences the function and structure of voltage gated calcium channel (VGCC) splice variants is poorly understood. Here we used long-read RNA-sequencing to catalog rat Cacna1e (Cav2.3) splice variants, and computationally prioritize which are likely to impact channel function. Result We sequenced Cacna1e transcripts from rat thalamus using Oxford Nanopore sequencing yielding the structure of 2,110 Cacna1e splice variants. Of these, up to 154 had the potential encode for a functional channel based on predicted amino acid sequences. Our analysis revealed a total of 31 cassette splicing events (in various combinations) potentially affecting channel function, with three cassette exons appreciably expressed and conserved. Conclusion Our work both provides the first long-read sequencing of Cacna1e and the first computational evaluation of Cacna1e splice variants for future follow-up. This overall strategy to provide the field with prioritized transcripts will improve our understanding of Cacna1e function, its role in disease pathophysiology, and serve as a general approach to evaluate splice variant function across multiple ion channel types.

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The Ca V 1.2 calcium channel is one primary pathway for Ca 2+ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular Ca V 1.2 functions under hyperglycemic condition remains unknown. Here, Sprague–Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K + -induced arterial constriction and Ca V 1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K + -induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and Ca V 1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, Ca V 1.2 channels exhibit hyperpolarized current–voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of Ca V 1.2 channels in VSMCs and MAs. Notably, GS increases K + -induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates Ca V 1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications.

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl⁻ channel activated by intracellular Ca2+ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5) P₂]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P₂ degradation, where TME-M16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P₂ depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P₂ sensitivity of both TME-M16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (P O). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P₂, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P₂. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P₂-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P₂, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

The activation of mitochondrial large conductance calcium-activated potassium (mitoBK Ca ) channels increases cell survival during ischemia/reperfusion injury of cardiac cells. The basic biophysical and pharmacological properties of mitoBK Ca correspond to the properties of the BK Ca channels from the plasma membrane. It has been suggested that the VEDEC splice variant of the KCNMA1 gene product encoding plasma membrane BK Ca is targeted toward mitochondria. However there has been no direct evidence that this protein forms a functional channel in mitochondria. In our study, we used HEK293T cells to express the VEDEC splice variant and observed channel activity in mitochondria using the mitoplast patch-clamp technique. For the first time, we found that transient expression with the VEDEC isoform resulted in channel activity with the conductance of 290 ± 3 pS. The channel was voltage-dependent and activated by calcium ions. Moreover, the activity of the channel was stimulated by the potassium channel opener NS11021 and inhibited by hemin and paxilline, which are known BK Ca channel blockers. Immunofluorescence experiments confirmed the partial colocalization of the channel within the mitochondria. From these results, we conclude that the VEDEC isoform of the BK Ca channel forms a functional channel in the inner mitochondrial membrane. Additionally, our data show that HEK293T cells are a promising experimental model for expression and electrophysiological studies of mitochondrial potassium channels.

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

Background L-type Ca 2+ channel Ca V 1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac Ca V 1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of Ca V 1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how Ca V 1.2 channels are alternatively spliced in diabetic heart remains unknown. Methods Diabetic rat models were established by using high-fat diet in combination with low dose streptozotocin. Cardiac function and morphology were assessed by echocardiography and HE staining, respectively. Isolated neonatal rat ventricular myocytes (NRVMs) were used as a cell-based model. Cardiac Ca V 1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca 2+ concentration was monitored by using Fluo-4 AM. Results We find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased Ca V 1.2 channel with alternative exon 9* (Ca V 1.2 E9* ), but unchanged that with alternative exon 8/8a or exon 33. The splicing factor Rbfox2 expression is also increased in diabetic heart, presumably because of dominate-negative (DN) isoform. Unexpectedly, high glucose cannot induce the aberrant expressions of Ca V 1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates Ca V 1.2 E9* channels proportion and downregulates Rbfox2 expression in NRVMs. By whole-cell patch clamp, we find GS application hyperpolarizes the current-voltage curve and window currents of cardiac Ca V 1.2 channels. Moreover, GS treatment raises K + -triggered intracellular Ca 2+ concentration ([Ca 2+ ] i ), enlarges cell surface area of NRVMs and induces hypertrophic genes transcription. Consistently, siRNA-mediated knockdown of Rbfox2 in NRVMs upregulates Ca V 1.2 E9* channel, shifts Ca V 1.2 window currents to hyperpolarization, increases [Ca 2+ ] i and induces cardiomyocyte hypertrophy. Conclusions AGEs, not glucose, dysregulates Rbfox2 which thereby increases Ca V 1.2 E9* channels and hyperpolarizes channel window currents. These make the channels open at greater negative potentials and lead to increased [Ca 2+ ] i in cardiomyocytes, and finally induce cardiomyocyte hypertrophy in diabetes. Our work elucidates the underlying mechanisms for Ca V 1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced Ca V 1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.