A Novel FRET-Based Assay Reveals 1:1 Stoichiometry of Apocalmodulin Binding Across Voltage-Gated Ca and Na Ion Channels

Abstract

Voltage-gated Ca and Na channels represent two important classes of ion channels where resident calmodulin (CaM) regulates channel gating, providing the cell with vital Ca2+ feedback. Functionally, a single CaM has been suggested to suffice for Ca2+-modulation of channel gating (Science, 304:432; JGP 131:197). However, in vitro structural and biochemical studies have argued that multiple Ca2+/CaMs may bind to the CaV channel carboxy-terminus (primary locus of CaM/channel interaction). Thus, the stoichiometry of apoCaM binding to the channel carboxy-terminus remains a crucial unknown. In this regard, live-cell FRET 2-hybrid assay has been used extensively to probe apoCaM binding. To gauge relative binding affinities, FRET efficiency between fluorophore-tagged apoCaM and a binding partner may be measured from acceptor-centric (33-FRET, Neuron39:97) or donor-centric (E-FRET, Biophys J91:L39) perspectives. Here, we exploit a fundamental asymmetry between these measures to estimate the stoichiometry of binding between apoCaM and channel carboxy termini: the ratio of maximal FRET efficiencies, as measured by 33-FRET and E-FRET methods, will approximate the ratio of donors to acceptors present in the complex. CFP and YFP concatemers with known ratios of donors and acceptors confirm this remarkable principle. Accordingly, we quantified both 33-FRET and E-FRET between CFP-apoCaM and YFP-tagged carboxy-terminal peptides of CaV1.3, CaV2.1, NaV1.4, and NaV1.5 channels. Remarkably, the maximal FRET ratios, computed via 33-FRET and E-FRET methods, equaled unity, arguing well for a 1:1 stoichiometry of apoCaM binding across CaV and NaV ion-channel families. These results mirror functional findings that a single CaM, pre-associated to the carboxy-termini of these channels, suffices for robust modulation of channel gating. More broadly, these experiments underscore the power of FRET 2-hybrid assay to discern multiple aspects of binding interactions within a live-cell context.