The formation and retention of distinct membrane domains in the fluidic membrane bilayer is the key process in establishing spatial organization for mediating physiological functions in metazoans. The spectrin-ankyrin network organizes diverse membrane domains including T-tubule and intercalated disc of cardiomyocytes, basolateral membrane of epithelial cells, costameres of striatal muscle, and axon initial segments and nodes of Ranvier in nervous system. This thesis identifies a novel function of 480 kDa ankyrin-G, an alternatively spliced isoform of the ankyrin family, in promoting somatodendritic GABAA receptor synaptogenesis both in vitro and in vivo. In the nervous system, an insertion of a neuronal specific exon (exon 37) occurs in ankyrin-G polypeptide which results in a 480 kDa isoform. 480 kDa ankyrin-G (giant ankyrin-G) has been shown to coordinate formation and maintenance of the axon initial segment (AIS) and nodes of Ranvier. This thesis research began with the discovery that giant ankyrin-G, previously thought to be confined to the axon initial segment, forms developmentally-regulated and cell-type specific somatodendritic ;;outposts;; on the plasma membrane of pyramidal neurons. This somatodendritic 480 kDa ankyrin-G outpost forms micron-scale membrane domains where it associates with canonical AIS binding partners including voltage-gated sodium channel and neurofascin. This thesis further discovered that the giant insert of 480 kDa ankyrin-G interacts with GABARAP, a GABAA receptor-associated protein. Both the interaction with GABARAP and the membrane association through palmitoylation of giant ankyrin-G are required for the formation of somatodendritic GABAergic synapses. This work further found that ankyrin-G associates with extrasynaptic GABAA receptors and stabilizes receptors on the extrasynaptic membrane through opposing endocytosis. This story demonstrates for the first time the existence of giant ankyrin-G somatodendritic outpost as well as its function in directing the formation of GABAergic synapses that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and neurodevelopmental disorders.
Additional work presented in the Appendix characterized novel ankyrin-G full length transcripts in the heart and kidney with unique domain compositions though alternative splicing. The preliminary work further identified biochemical properties and potential role of an insert C in the C-terminus of ankyrin-G in mediating cytokinesis and cellular migration in mouse fibroblasts. Together, this thesis work expands the knowledge of giant ankyrin-G functions in the nervous system and offers insights into the diversified roles of distinct ankyrin-G peptides acquired from alternative splicing in organizing specific membrane domains and interacting with defined intracellular pathways in different tissues.
