Figure 6: Binding of CaM G114R to CaV1.3 is partially restored by elevated Ca2+.
(A-B) FRET binding curves from a live-cell flow cytometric FRET assay for the CaV1.3 channel fragment and CaM WT (A) or CaM G114R (B) performed in high Ca2+ conditions. Each point reflects the result from an individual cell, with a curve in red fitted to estimate effective KD (KD,eff). Low (basal) Ca2+ results are reproduced as the gray dashed curve for reference. C: Effective KD values (KD,eff) plotted for the CaV1.3 channel fragments, CaM WT, and CaM G114R under conditions of minimal (grey) or elevated cytosolic Ca2+ (red). (****, p > 0.0001 compared to WT in high Ca2+; #, p = 0.029 compared to apoCaM G114R). Data plotted ± SEM. (D) Proposed mechanism under which endogenous WT CaM (grey) is capable of binding to CaV1.3 and conferring CDI in the presence or absence of nonbinding CaM G114R (orange). In CaV1.3L with the ICDI/CTM (red) present, CDI occurs only when CaM is able to displace ICDI/CTM. Under endogenous expression conditions, CaM expression levels are not sufficient to dislodge ICDI/CTM for the majority of channels, resulting in a reduction in CDI. Overexpression of CaM G114R does not significantly alter this scenario as it cannot compete with ICDI/CTM, however overexpression of CaM WT enhances CDI compared to endogenous CaM via displacement of ICDI/CTM. In contrast, CaM WT, either endogenous or overexpressed, confers robust CDI in the absence of ICDI/CTM in CaV1.343S. Exemplar data above and below is reproduced from Figs. 2, 4 and from (Sang et al., 2021) for CaV1.343s (endogenous CaM) and from (Limpitikul et al., 2016) for CaV1.3L (endogenous CaM) for comparison.