Selective posttranslational inhibition of Ca(V)β(1)-associated voltage-dependent calcium channels with a functionalized nanobody

Ca(2+) influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca(2+) influx, to mediate disparate functions is diversity of HVACC pore-forming α(1) and auxiliary Ca(V)β(1)–Ca(V)β(4) subunits. Selective Ca(V)α(1) blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on Ca(V)β 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(V)β isoforms. Here, we identify a nanobody (nb.E8) that selectively binds Ca(V)β(1) SH3 domain and inhibits Ca(V)β(1)-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(V)β(1)-reconstituted Ca(V)1/Ca(V)2 and native Ca(V)1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca(2+) influx and excitation-transcription coupling in hippocampal neurons, but was inert against Ca(V)β(2)-associated Ca(V)1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of Ca(V)β(1) signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.