Project Summary: Proper development of the human cortex is essential for brain function and depends on the synchronization of complex molecular and cellular processes. Malformations of cortical development may occur in the setting of genetic mutations altering the activity of genes essential for this synchrony. Polymicrogyria (PMG) is a malformation of cortical development characterized by abnormal folding of the cerebral cortex and is often associated with epilepsy and intellectual disability. Our lab has performed whole exome sequencing (WES) of over 200 families affected with PMG and no previously identified diagnosis, establishing the genetic landscape of PMG pathogenesis. From our cohort, nearly 20% of mutations found in solved cases occur in ion channels with high in utero expression, implicating developmental channelopathies as a key pathway in PMG pathogenesis. However, the role of ion channels in cortical histogenesis—and their contribution to disease— remains poorly understood. Through our exome analysis, we identified three affected individuals with de novo missense variants in the gene PANX1, encoding Pannexin 1. PANX1 forms a heptameric ion channel of seven PANX1 subunits and is expressed in early development in neurons and glia. Opening of the heptameric ion channel releases small anions and ATP into the extracellular milieu, participating in purinergic signaling. The channel is further speculated to contribute to the propagation of calcium waves—a form of developmental electrical signaling that drives cell proliferation and migration—yet its definitive contribution in the fetal cortex is unknown. Thus, in this proposal, I aim to 1) characterize our novel missense PANX1 variants and their impact on channel activity and 2) channel structure, and 3) leverage the gyrencephalic ferret as an animal model to study PANX1’s role in cortical development. In Aim 1, I will investigate how our newly identified PMG-associated PANX1 variants alter protein function. By overexpressing mutant constructs in heterologous cells, I will assess expression and localization using western blotting. Furthermore, using both heterologous cells and induced pluripotent stem (iPSC)-derived neurons, I will determine alterations to ATP release and channel conductance with patch clamping. In Aim 2, I will perform cryo-electron microscopy on purified wildtype and mutant channels, assessing how mutations alter structural properties and gap junction formation. These two aims will provide a molecular, cellular, and structural level mechanism for PANX1 perturbation in disease. In Aim 3, I will provide functional evidence for the association between PANX1 activity and cortical histogenesis in ferrets. While mice are lissencephalic, ferrets develop gyri, enabling investigation of disordered gyrification. I will also use calcium imaging to characterize the circuit level impact of PANX1 disruption on developmental calcium signaling. Functional investigation of human PANX1 va...