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

Ca influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca influx, to mediate disparate functions is diversity of HVACC pore-forming α and auxiliary Caβ-Caβ subunits. Selective Caα blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on Caβ isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of Caβ isoforms. Here, we identify a nanobody (nb.E8) that selectively binds Caβ SH3 domain and inhibits Caβ-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 Caβ-reconstituted Ca1/Ca2 and native Ca1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca influx and excitation-transcription coupling in hippocampal neurons, but was inert against Caβ-associated Ca1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of Caβ signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.