Abstract
Inhibiting high-voltage-activated calcium channels (HVACCs; CaV1/CaV2) is therapeutic for myriad cardiovascular and neurological diseases. For particular applications, genetically-encoded HVACC blockers may enable channel inhibition with greater tissue-specificity and versatility than is achievable with small molecules. Here, we engineered a genetically-encoded HVACC inhibitor by first isolating an immunized llama nanobody (nb.F3) that binds auxiliary HVACC CaVβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to CaVβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (CaV-aβlator), an E3 ubiquitin ligase, ablated currents from diverse HVACCs reconstituted in HEK293 cells, and from endogenous CaV1/CaV2 channels in mammalian cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, CaV-aβlator redistributed CaV1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces CaV-aβlator as a potent genetically-encoded HVACC inhibitor, and describes a general approach that can be broadly adapted to generate versatile modulators for macro-molecular membrane protein complexes.
Highlights
Inhibition of high-voltage-activated calcium channels (HVACCs) is an important prevailing or potential therapy for diverse cardiovascular and neurological diseases (Zamponi et al, 2015)
We identified at least seven distinct classes of nanobody binders based on the unique sequences within complementarity determining regions (CDR1-3), the major determinants of antigen binding (Figure 1d,e)
Similar experiments conducted with the other CaVbs (b2-b4) showed that they all bind with nb.F3-cyan fluorescent protein (CFP)-C1 in cells (Figure 1f; Figure 1—figure supplement 1), indicating the nanobody interacts with an epitope conserved among CaVbs
Summary
Inhibition of high-voltage-activated calcium channels (HVACCs) is an important prevailing or potential therapy for diverse cardiovascular (hypertension, cardiac arrhythmias, cerebral vasospasm) and neurological diseases (epilepsy, chronic pain, Parkinson’s disease) (Zamponi et al, 2015). Rad/Rem/Rem2/Gem/Kir (RGK) proteins are endogenous small Ras-like G-proteins that profoundly inhibit all HVACCs when over-expressed in either heterologous cells or native tissue (Beguin et al, 2001; Finlin et al, 2003; Chen et al, 2005; Xu et al, 2010) They form ternary complexes with HVACCs via binding to constituent b subunits and inhibit currents via multiple mechanisms including removal of surface channels and impairing gating (Yang and Colecraft, 2013; Yang et al, 2010). The resulting construct, termed CaV-ablator, eliminated diverse HVACCs both in both reconstituted systems and native excitable cells, providing a unique new tool for probing CaV1/CaV2 signaling and regulation in vivo, and potential development into a therapeutic
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