A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits

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.

Designer genetically encoded voltage‐dependent calcium channel inhibitors inspired by RGK GTPases

“…One of these nanobodies, termed nb.F3, bound all four Ca V β isoforms when expressed in cells (Morgenstern et al . 2019), which was not surprising given the high homology among these auxiliary subunits in their conserved src homology 3 (SH3) and guanylate kinase (GK) domains (Chen et al . 2004; Opatowsky et al .…”

“…3 and ) (Morgenstern et al . 2019). Whole‐cell patch clamp experiments demonstrated that nb.F3‐Nedd4L essentially eliminated reconstituted Ca V 1.2, Ca V 1.3 and Ca V 2.1‐Ca V 2.3 channel currents (Fig.…”

“…Ca V ‐ablator also proved effective in eliminating endogenous Ca V 1/Ca V 2 channels in pancreatic β‐cells, dorsal root ganglion (DRG) neurons and cardiac myocytes (Morgenstern et al . 2019). Examination of how Ca V ‐ablator eliminated Ca V 1.2 currents in ventricular cardiomyocytes indicated that pore‐forming α 1C subunits were re‐directed from dyadic junctions to intracellular compartments, specifically Rab 7‐positive late endosomes (Morgenstern et al .…”

“…Examination of how Ca V ‐ablator eliminated Ca V 1.2 currents in ventricular cardiomyocytes indicated that pore‐forming α 1C subunits were re‐directed from dyadic junctions to intracellular compartments, specifically Rab 7‐positive late endosomes (Morgenstern et al . 2019).…”

Estudio del efecto del receptor de ghrelina (GHSR) sobre la densidad en membrana de canales de calcio operados por voltaje (CaV) y sobre las propiedades biofísicas del sub-tipo CaV3

“…En relación a NeDD4, recientemente Morgenstern y colaboradores han diseñado un inhibidor recombinante en base a dicha proteína partiendo de la interacción entre Ca V β y NeDD4 reportada previamente. Con dicho inhibidor han logrado impedir la interacción física entre las subunidades Ca V α 1 de los HVA en forma selectiva y consecuentemente la funcionalidad de dichos canales (Morgenstern et al, 2019). Nuevos experimentos son necesarios para concluir acerca de los efectores moleculares que median el efecto de la actividad constitutiva de GHSR sobre el tráfico de los Ca V.…”