Figure 4. Stoichiometry of calmodulin binding to Ca_V and Na_V channels.

(a) Cartoon illustrates FRET pairs ECFP-CaM and Ca_V1.2 holochannel tagged with EYFP on its carboxy-terminus. The Ca_V channel auxiliary subunits β_2A and α₂δ subunits are coexpressed. (b) Bar-graph summary of maximal 3³-FRET (E_A,max; black) and E-FRET (E_D,max; red) efficiencies under basal (– Ca²⁺) and elevated Ca²⁺ conditions for CaM binding to Ca_V1.2 (mean±s.e.m.; n, number of cells, as labelled for each bar). E_A,max and E_D,max are approximately equal under resting Ca²⁺ conditions. With high cytosolic Ca²⁺ levels, E_A,max∼2 × higher than E_D,max. (c) Computing stoichiometry ratio (ν) shows that a single CaM binds to the holo-Ca²⁺ channels under low Ca²⁺ conditions while two CaM interact with the channel complex upon Ca²⁺ elevation (mean±s.e.m.). (d) Cartoon depicts FRET pairs ECFP-CaM and Na_V1.4 with EYFP fused to its carboxy-terminus. (e) Bar-graph summarizes maximal 3³-FRET (E_A,max; black) and E-FRET (E_D,max; red) efficiencies for CaM binding to Na_V1.4 (mean±s.e.m.; n, number of cells, as labelled for each bar). Notice that maximal 3³-FRET and E-FRET efficiencies are approximately equal to each when measured under both basal and elevated Ca²⁺ conditions. (f) Experimentally determined stoichiometry ratio (ν) shows that a single CaM interacts with Na_V channel complex under all Ca²⁺ conditions. Format as in c.