Ca2+ influx through high-voltage-gated Ca2+ channels (HVGCCs) is necessary for converting electrical signals into physiological responses in excitable cells, and mediates diverse functions including controlling neurotransmitter release, tuning neuronal excitability, and coupling electrical activity to regulation of gene expression. Structurally, HVGCCs are hetero-multimeric macromolecular complexes comprised of a pore-forming α1 polypeptide assembled with auxiliary (β, α2δ, and in some cases γ) subunits. There are four CaVβ subunit isoforms (β1-β4) encoded by distinct genes – CACNB1–CACNB4. CaVβs are cytosolic proteins that are necessary for trafficking α1 subunits to the plasma membrane and also regulate various aspects of channel gating (voltage-dependence and kinetics of activation and inactivation; and open probability, Po). Somatosensory neurons express multiple HVGCC pore-forming α1 (CaV1.2, CaV2.1, CaV2.2, and CaV2.3) and β (β1-β4) subunit isoforms. Nerve injury leads to significantly decreased HVGCC currents even though expression of CaVα1 subunits remain unchanged. By contrast, β3 and β4 (but not β1 and β2) expression declines in some populations of dorsal root ganglion (DRG) neurons after nerve injury, potentially underlying the diminished whole-cell Ca2+ current (ICa) and contributing to pathology. While the functional roles of distinct CaVα1 subunits are readily studied using available small molecule and toxin blockers, the functional logic of CaVβ molecular diversity is understudied and underappreciated due to a lack of tools capable of post-translationally inhibiting HVGCCs based on the identity of the CaVβ isoform associated with them. In previous work, we pioneered a unique targeted ubiquitination approach that effectively removes HVGCC complexes from the cell surface by linking a nanobody (nb) that non-selectively binds all auxiliary CaVβs to the catalytic HECT domain of the E3 ubiquitin ligase, NEDD4L (termed CaV-aβlator). By contrast to shRNA knockdown or gene knockout approaches, CaV-aβlator post-translationally inhibits the entire channel complex (rather than remove just the targeted β subunit) limiting confounding interpretations due to molecular reshuffling or overlapping functions of distinct CaVβ isoforms. The overall objectives of this proposal are to: 1) develop a protein engineering approach for the post-translational inhibition of HVGCC complexes based on the identity of the associated CaVβ-isoform, utilizing the principle of targeted ubiquitination, and 2) exploit newly created genetically-encoded inhibitors to probe the functional logic of CaVβ molecular diversity in DRG neurons. We propose two specific Aims: (1) Develop genetically-encoded CaVβ-isoform specific HVGCC inhibitors (Chisels). (2) Utilize engineered CaVβ-targeted nanobodies to probe the functional logic of CaVβ molecular diversity in somatosensory neurons. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to understand the basis of pain and enhance clinical pain management.

NARRATIVE The proposal seeks to develop novel, protein inhibitors of voltage-gated calcium channels by targeting their auxiliary β subunit isoforms. The results will enable new ways of probing the roles of calcium channels in pain and addiction pathways.