Calmodulation of Voltage-Gated Calcium Channels by Blue Light

Abstract

Delivering custom-shaped signaling inputs to dissect biological networks has been a coveted goal of engineering-minded biologists, keenly aware of the advantages of analyzing electronic circuitry with a signal generator. We have recently devised a rapamycin-based chemical-dimerization strategy to produce step-like increases in the concentration of calmodulin (CaM) at the cytoplasmic mouth of L-type Ca2+ channels. This tactic revealed that CaM binding to channels induces two effects: a large increase in peak open probability (PO), and the ability to undergo Ca2+-dependent inactivation (CDI) (Adams et al (2014) Cell, in press). Yet, the rapamycin system is comparatively slow (trise-half ∼30 s) and irreversible. Here we explore the use of a light-based dimerization system from Arabidopsis (CRY2 and CIB1, Kennedy et al (2010)) to deliver CaM more rapidly and reversibly upon activation by blue light. To evaluate functionality with Ca2+ channels, HEK293 cells expressing Ltype Cav1.3 channels were endowed with CIB1 targeted to the membrane and CRY2 glycine linked to a dominant-negative CaM1234, where mutations eliminate Ca2+ binding and presumably render this construct a constitutive apoCaM. On dimerization with blue light, CDI was indeed attenuated, confirming functionality with Ca2+ channels. More telling, blue-light exposure left peak currents unchanged, arguing that CaM1234 in fact acts just like apoCaM. Next, we expressed a variant of Cav1.3 (MQDY) with diminished apoCaM binding at baseline, and changed the CIB1 payload to wild-type CaM. Reassuringly, blue-light exposure increased both peak current and CDI by ∼20% (5/6 responding cells), consistent with enhanced MQDY binding to CaM upon blue-light recruitment. Intriguingly, full enhancement occured in the very first current evoked after blue-light illumination (<20 s); CaM exchange with these channels thereby occurs on an even faster timescale. In all, light-based recruitment promises a powerful tool for investigating CaM modulation of membrane proteins.