Ankyrin-B p.S646F undergoes increased proteasome degradation and reduces cell viability in the H9c2 rat ventricular cardiomyoblast cell line.

Ankyrin-B (AnkB) is scaffolding protein that anchors integral membrane proteins to the cardiomyocyte cytoskeleton. We recently identified an AnkB variant, AnkB p.S646F (ANK2 c.1937 C>T) associated with a phenotype ranging from predisposition for cardiac arrhythmia to cardiomyopathy. AnkB p.S646F exhibited reduced expression levels in the H9c2 rat ventricular-derived cardiomyoblast cell line relative to wildtype AnkB. Here, we demonstrate that AnkB is regulated by proteasomal degradation and proteasome inhibition rescues AnkB p.S646F expression levels in H9c2 cells, although this effect is not conserved with differentiation. We also compared the impact of wildtype AnkB and AnkB p.S646F on cell viability and proliferation. AnkB p.S646F expression resulted in decreased cell viability at 30 h after transfection, whereas we observed a greater proportion of cycling, Ki67-positive cells at 48 h after transfection. Notably, the number of GFP-positive cells was low and was consistent between wildtype AnkB and AnkB p.S646F expressing cells, suggesting that AnkB and AnkB p.S646F affected paracrine communication between H9c2 cells differentially. This work reveals that AnkB levels are regulated by the proteasome and that AnkB p.S646F compromises cell viability. Together, these findings provide key new insights into the putative cellular and molecular mechanisms of AnkB-related cardiac disease.

Ankyrin B and Ankyrin B variants differentially modulate intracellular and surface Cav2.1 levels.

Ankyrin B (AnkB) is an adaptor and scaffold for motor proteins and various ion channels that is ubiquitously expressed, including in the brain. AnkB has been associated with neurological disorders such as epilepsy and autism spectrum disorder, but understanding of the underlying mechanisms is limited. Cav2.1, the pore-forming subunit of P/Q type voltage gated calcium channels, is a known interactor of AnkB and plays a crucial role in neuronal function. Here we report that wildtype AnkB increased overall Cav2.1 levels without impacting surface Cav2.1 levels in HEK293T cells. An AnkB variant, p.S646F, which we recently discovered to be associated with seizures, further increased overall Cav2.1 levels, again with no impact on surface Cav2.1 levels. AnkB p.Q879R, on the other hand, increased surface Cav2.1 levels in the presence of accessory subunits α2δ1 and ß4. Additionally, AnkB p.E1458G decreased surface Cav2.1 irrespective of the presence of accessory subunits. In addition, we found that partial deletion of AnkB in cortex resulted in a decrease in overall Cav2.1 levels, with no change to the levels of Cav2.1 detected in synaptosome fractions. Our work suggests that depending on the particular variant, AnkB regulates intracellular and surface Cav2.1. Notably, expression of the AnkB variant associated with seizure (AnkB p.S646F) caused further increase in intracellular Cav2.1 levels above that of even wildtype AnkB. These novel findings have important implications for understanding the role of AnkB and Cav2.1 in the regulation of neuronal function in health and disease.

Pannexin 1 Regulates Network Ensembles and Dendritic Spine Development in Cortical Neurons.

Dendritic spines are the postsynaptic targets of excitatory synaptic inputs that undergo extensive proliferation and maturation during the first postnatal month in mice. However, our understanding of the molecular mechanisms that regulate spines during this critical period is limited. Previous work has shown that pannexin 1 (Panx1) regulates neurite growth and synaptic plasticity. We therefore investigated the impact of global Panx1 KO on spontaneous cortical neuron activity using Ca2+ imaging and in silico network analysis. Panx1 KO increased both the number and size of spontaneous co-active cortical neuron network ensembles. To understand the basis for these findings, we investigated Panx1 expression in postnatal synaptosome preparations from early postnatal mouse cortex. Between 2 and 4 postnatal weeks, we observed a precipitous drop in cortical synaptosome protein levels of Panx1, suggesting it regulates synapse proliferation and/or maturation. At the same time points, we observed significant enrichment of the excitatory postsynaptic density proteins PSD-95, GluA1, and GluN2a in cortical synaptosomes from global Panx1 knock-out mice. Ex vivo analysis of pyramidal neuron structure in somatosensory cortex revealed a consistent increase in dendritic spine densities in both male and female Panx1 KO mice. Similar findings were observed in an excitatory neuron-specific Panx1 KO line (Emx1-Cre driven; Panx1 cKOE) and in primary Panx1 KO cortical neurons cultured in vitro. Altogether, our study suggests that Panx1 negatively regulates cortical dendritic spine development.

Probenecid Disrupts a Novel Pannexin 1-Collapsin Response Mediator Protein 2 Interaction and Increases Microtubule Stability.

Neurite formation relies on finely-tuned control of the cytoskeleton. Here we identified a novel protein-protein interaction between the ion and metabolite channel protein Pannexin 1 (Panx1) and collapsin response mediator protein 2 (Crmp2), a positive regulator of microtubule polymerization and stabilization. Panx1 and Crmp2 co-precipitated from both Neuro-2a (N2a) cells and mouse ventricular zone (VZ) tissue. In vitro binding assays between purified proteins revealed the interaction occurs directly between the Panx1 C-terminus (Panx1 CT) and Crmp2. Because Crmp2 is a well-established microtubule-stabilizing protein, and we previously observed a marked increase in neurite formation following treatment with the Panx1 blocker, probenecid, in N2a cells and VZ neural precursor cells (NPCs), we investigated the impact of probenecid on the Panx1-Crmp2 interaction. Probenecid treatment significantly disrupted the Panx1-Crmp2 interaction by both immunoprecipitation (IP) and proximity ligation analysis, without altering overall Crmp2 protein expression levels. In the presence of probenecid, Crmp2 was concentrated at the distal ends of growing neurites. Moreover, probenecid treatment increased tubulin polymerization and microtubule stability in N2a cells. These results reveal that probenecid disrupts a novel interaction between Panx1 and the microtubule stabilizer, Crmp2, and also increases microtubule stability.