Selective posttranslational inhibition of Ca_Vβ₁-associated voltage-dependent calcium channels with a functionalized...

Ca²⁺ influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca²⁺ influx, to mediate disparate functions is diversity of HVACC pore-forming α₁ and auxiliary Ca_Vβ₁-Ca_Vβ₄ subunits. Selective Ca_Vα₁ blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on Ca_Vβ isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of Ca_Vβ isoforms. Here, we identify a nanobody (nb.E8) that selectively binds Ca_Vβ₁ SH3 domain and inhibits Ca_Vβ₁-associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through Ca_Vβ₁-reconstituted Ca_V1/Ca_V2 and native Ca_V1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca²⁺ influx and excitation-transcription coupling in hippocampal neurons, but was inert against Ca_Vβ₂-associated Ca_V1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of Ca_Vβ₁ signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.