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

ABSTRACT: Inhibiting high-voltage-activated calcium channels (HVACCs; Ca_V1/Ca_V2) 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 Ca_Vβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to Ca_Vβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (Ca_V-aβlator), an E3 ubiquitin ligase, ablated currents from diverse HVACCs reconstituted in HEK293 cells, and from endogenous Ca_V1/Ca_V2 channels in mammalian cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, Ca_V-aβlator redistributed Ca_V1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces Ca_V-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.

SUBMITTER: Morgenstern TJ

PROVIDER: S-EPMC6701945 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As with other elements of the peripheral auditory system, spiral ganglion neurons display specializations that vary as a function of location along the tonotopic axis. Previous work has shown that voltage-gated K(+) channels and synaptic proteins show graded changes in their density that confers rapid responsiveness to neurons in the high frequency, basal region of the cochlea and slower, more maintained responsiveness to neurons in the low frequency, apical region of the cochlea. In order to understand how voltage-gated calcium channels (VGCCs) may contribute to these diverse phenotypes, we identified the VGCC α-subunits expressed in the ganglion, investigated aspects of Ca(2+)-dependent neuronal firing patterns, and mapped the intracellular and intercellular distributions of seven VGCC α-subunits in the spiral ganglion in vitro. Initial experiments with qRT-PCR showed that eight of the ten known VGCC α-subunits were expressed in the ganglion and electrophysiological analysis revealed firing patterns that were consistent with the presence of both LVA and HVA Ca(2+) channels. Moreover, we were able to study seven of the α-subunits with immunocytochemistry, and we found that all were present in spiral ganglion neurons, three of which were neuron-specific (Ca(V)1.3, Ca(V)2.2, and Ca(V)3.3). Further characterization of neuron-specific α-subunits showed that Ca(V)1.3 and Ca(V)3.3 were tonotopically-distributed, whereas Ca(V)2.2 was uniformly distributed in apical and basal neurons. Multiple VGCC α-subunits were also immunolocalized to Schwann cells, having distinct intracellular localizations, and, significantly, appearing to distinguish putative compact (Ca(V)2.3, Ca(V)3.1) from loose (Ca(V)1.2) myelin. Electrophysiological evaluation of spiral ganglion neurons in the presence of TEA revealed Ca(2+) plateau potentials with slopes that varied proportionately with the cochlear region from which neurons were isolated. Because afterhyperpolarizations were minimal or absent under these conditions, we hypothesize that differential density and/or kinetics of one or more of the VGCC α-subunits could account for observed tonotopic differences. These experiments have set the stage for defining the clear multiplicity of functional control in neurons and Schwann cells of the spiral ganglion.

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

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Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

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