SUMMARY CACNA2D1 – CACNA2D4 genes encode alpha2delta-1, alpha2delta-2, alpha2delta-3, and alpha2delta-4 proteins which are most well known as auxiliary subunits of high-voltage-activated Ca2+ channels. There are seven different HVA CaV channel types (CaV1.1 – CaV1.4; CaV2.1 – CaV2.3), and in general, alpha2delta-1 – alpha2delta-4 proteins can interact promiscuously with any of the seven pore-forming subunits to enhance trafficking and stabilize channels at the cell surface to increase whole-cell current. Nevertheless, these proteins have broad but not completely overlapping distributions, and loss-of-function mutations in humans, or their individual knockout in animal models, gives rise to distinct disease phenotypes, indicating some unique functional properties. Missense mutations or knockout of alpha2delta-1 and alpha2delta-2 are most prominently linked to neurological symptoms including epilepsy and intellectual disability; loss of alpha2delta-3 is associated with malignancies including gliomas and nasopharyngeal carcinoma; and dysregulation of alpha2delta-4 causes retinal dystrophy and night blindness. The potential of this protein family as therapeutic targets to treat disease was elevated by the serendipitous finding that gabapentinoids which are used clinically to treat neuropathic pain and epilepsy exert their actions via binding alpha2delta-1. It is less clear precisely how gabapentin binding to alpha2delta-1 alleviates neuropathic pain. Proposed candidate mechanisms include chronic down-regulation of CaV2 channel trafficking; disruption of alpha2delta-1 interaction with extracellular thrombospondins; and prevention of alpha2delta-1 mediated trafficking of NMDA type glutamate receptors to the synaptic terminus in the dorsal horn. Thus, beyond their role as auxiliary CaV channel subunits, alpha2delta subunits may also differentially interact with other proteins which may play a role in their distinctive physiological functions. This proposal is primarily focused on CACNA2D2 and CACNA2D3 which are two genes included in the list of IDG-eligible understudied proteins. Ligands that can specifically interact with these proteins and be used to either detect them in situ or manipulate their function are essential for illuminating their specific functional roles as well as realizing their potential as therapeutic targets. Here, we propose to develop nanobodies to alpha2delta-2 and alpha2delta-3 as novel reagents to detect and manipulate the functions of these proteins in vitro, in situ, and in vivo. Our lab has recently developed nanobodies against auxiliary CaV? subunits and used these genetically-encoded molecules to regulate functional expression of CaV1/CaV2 channels with unprecedented levels of precision. The expertise we have gained from these previous studies not only demonstrate overall feasibility, but also provides preliminary data that bolsters our expectations that the experiments proposed here will yield unique reagents to illuminate alpha2delta-2 and alpha2delta-3 biology and therapeutic potential. (1) Isolate nanobodies against alpha2delta-2 and alpha2delta-3 and characterize their binding characteristics. (2) Determine functional impact of alpha2delta-2 and alpha2delta-3 nanobodies on CaV channel functional expression. (3) Determine impact of alpha2delta-2/alpha2delta-3 nanobodies on putative non-CaV channel functions of alpha2delta subunits.

NARRATIVE The proposal seeks to develop novel nanobody reagents to detect and manipulate function of two understudied proteins, alpha2delta-2 and alpha2delta-3, respectively encoded by CACNA2D2 and CACNA2D3 genes.