Preassociation of calmodulin with voltage-gated Ca²⁺ channels revealed by FRET in single living cells

Among the most intriguing forms of Ca²⁺ channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca²⁺, acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca²⁺-insensitive mutant CaM abolishes Ca²⁺-dependent modulation, hinting that Ca²⁺-free CaM may "preassociate" with these channels to enhance detection of local Ca²⁺. Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. Here, we develop a three filter-cube fluorescence resonance energy transfer method (three-cube FRET) to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca²⁺-independent associations between CaM and the pore-forming α₁ subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions.