Ca2+ Sensitivity of L-Type Calcium Channel Inactivation Probed by Ca2+ Photouncaging--- Window onto Calmodulinopathies

Abstract

Mutations in calmodulin (CaM) cause long-QT syndrome and recurrent cardiac arrest (Circulation (2013) 127: 1009). Arrhythmogenesis likely arises from impaired CaM regulation of ion channels, especially Ca2+-dependent inactivation (CDI) of L-type calcium channels (LTCCs). Yet, traditional assessment of the Ca2+ sensitivity of CDI conflates channel gating (controlling Ca2+ influx), and actual Ca2+ responsiveness of CDI. Here, we used Ca2+-photouncaging to deliver known Ca2+ steps to LTCCs. Li+ was the charge carrier to restrict the source of Ca2+ to that uniformly photouncaged by UV flashes. Panel A displays the CaM C-lobe component of CDI, isolated by coexpressing LTCCs with mutant CaM12 (Ca2+ binding only to C-lobe). Absent Ca2+ uncaging, quasi-steady currents were evoked (black). Ca2+ uncaging induced strong and kinetically resolved CDI (gray, with fit). Isolating the N-lobe form of CDI (panel B) yielded a slower but also strong form of CDI. Data such as these enabled full profiles of steady-state and kinetic responsiveness of CDI to Ca2+. Intriguingly, disease-related CaM mutations resulted in well-resolved and specific deficits in the Ca2+-to-CDI response profile, offering powerful insight into the channel mechanistic alterations that ultimately yield system-level disease.View Large Image | View Hi-Res Image | Download PowerPoint Slide