Authors :
Carina Elvira, BS – University of Michigan Medical School
Kalynn Harvey, BS – University of Michigan Medical School
Gracie Kmiec, BS – University of Michigan Medical School
Lia Min, PhD – University of Michigan Medical School
Kevin Bender, PhD – University of California, San Francisco
Presenting Author: Paul Jenkins, PhD – University of Michigan Medical School
Rationale:
Sodium channels (NaVs) and their primary scaffolding partners, ankyrins, are central to neuronal excitability. Dysfunction in these proteins and their interactions are major risk factors for neurodevelopmental disorders, including developmental and epileptic encephalopathies, Dravet syndrome, intellectual disability, and autism spectrum disorder. NaVs are localized to specialized excitable membrane domains, including the axon initial segment and nodes of Ranvier, by ankyrin-G (ANK3), a member of the ankyrin family of intracellular scaffolding proteins. Recently, we showed that another ankyrin family member, ankyrin-B (ANK2) scaffolds NaV1.2 (SCN2A) in dendrites of neocortical pyramidal cells, where these channels mediate action potential backpropagation, a process necessary for normal neuronal excitability and synaptic plasticity. While key domains necessary for interactions between ankyrins and NaVs have been previously identified, these domains are often highly conserved, if not identical, between different members of the NaV and ankyrin families. Despite this, NaVs and ankyrins display unique subcellular localization and function, often differing between cell types, raising the important question of how these interactions are regulated.
Methods:
To address this question, we use a combination of protein biochemistry, cellular and molecular biology approaches, and electrophysiology to understand how ankyrin-NaV interactions are regulated. We also utilize cultured cells (HEK293-T) and rodent neuronal cultures to determine the effects of manipulating these mechanisms on channel localization and function.
Results:
Previous studies have identified the intracellular loop between the second and third sets of transmembrane domains (II-III loop) on NaVs as the primary determinant of interaction with ankyrins because of a conserved nine amino acid motif within this loop. Here, we examine the sequence determinants within the loop and effects of posttranslational modifications channel localization and function. We find that, like NaV1.6, NaV1.2 is modified by the 16-carbon fatty acid posttranslational modification palmitoylation and that disruption of the palmitoylation sites within the II-III loop affects both channel biophysical properties and localization. We also show that the II-III loop is insufficient to explain the differences in subcellular localization between NaV1.2 and Nav1.6.
Conclusions:
These results are important for understanding how the subcellular localization of different members of the Nav1 family is controlled during normal neurodevelopment and in specific neuron types. Manipulation of these pathways could provide unique strategies for altering NaV localization and function.
Funding:
This work was supported by the NIH R01MH126960 (PMJ), R01MH126960-S1 (CCE), and T32GM007767 (KMH and GEK).