The long term objectives of this project are to explain the basis for the complex genotype-phenotype relationships for a growing number of severe calcium channel gene disorders and develop therapies based on these new insights. To make these advances we will capitalize on our new discovery that at least three of these calcium channel genes are bicistronic i.e. they encode two distinct proteins, the calcium channel proteins and a newly discovered transcription factor. We have discovered that the transcription factor is translated by internal translation by a process resembling an internal ribosomal entry site (IRES). We hypothesize that mutations in these Ca2+ channel genes may have a diversity of outcomes, affecting, in distinct cases, neuronal firing, calcium signaling, regulation of the expression of the transcription factor or directly altering the function of the transcription factor. In this study we will systematically explore the function and biological action of these three novel transcription factors, how their expression is regulated by the IRES, how normal cellular physiology governs their translocation to and from the nucleus, and how different mutations affecting channel gating, IRES function and transcription factor cause impaired neuronal development and or viability in these different disorders. We will study this using recombinant calcium channels expressed in primary neurons and human reprogrammed neurons from normal and patient sources, and in transgenic mice expressing well characterized mutations. We will study gene binding and expression using next generation approaches, nuclear translocation using epitope and fluorescent tags with physiological stimuli, IRES function using dual luciferase reporters and immunoblotting, neuronal development using immunofluorescent microscopy and corrective therapy using antisense oligos, miRNA and AAV viral vectors expressing transcription factors.
With this research we hope to untangle the complexities and challenges that have been encountered when trying to correlate the vast number of new mutations in discovered in calcium channel genes in individuals with diverse and overlapping neurological and neuropsychiatric symptoms so we can develop treatments. We will take advantage of a new discovery we have made that these genes produce not one, but two distinct proteins, the calcium channel protein and a new protein called a transcription factor. We will determine how the two proteins function together, how, with different mutations, this cooperation breaks down, and how to develop therapies by correcting the breakdown in integration.
