Figure 2. PKA regulation of the interaction between IQ and ICDI domains in Ca_V1.4.

(a) PKA regulation of L-type Ca²⁺ channel calmodulation. Left: ICDI module (red rectangle) on the DCT of the channel interacts with the IQ domain (pink circle) and dislodges CaM (green dumbbell). Without CaM, channels have low P_O and minimal CDI (idealized current underneath the cartoon). Right: PKA phosphorylation (orange lollipop) could weaken the IQ/ICDI interaction and allow CaM to rebind, thus increasing channel P_O and CDI. (b) Diagram of the C-tail of the channel illustrating the relevant FRET pairs, Venus-tagged IQ-A domains and Cerulean-tagged ICDI domain from Ca_V1.4. (c) FRET binding curve for Venus-IQ-A_1.4 paired with Cerulean-ICDI_1.4 in aGPVMs (grey). Isoproterenol (0.5 μM iso; red) decreases the relative binding affinity. Each point indicates a single cell. The control binding curve is replicated as the grey curve in the right-hand panel here and throughout. FR, FRET ratio; D_free, relative concentration of unbound Cerulean-tagged ICDI_1.4. (d) Kinetics of normalized fluorescent signals from YFP channel (S_YFP), CFP channel (S_CFP), FRET channel (S_FRET) and calculated FR from an exemplar aGPVM expressing Venus-IQ-A_1.4 and Cerulean-ICDI_1.4, with 0.5 μM isoproterenol and 10 μM H89 added as indicated. Fluorescent signals were normalized to baseline. (e) FRET binding curve for Venus-IQ-A_1.4 paired with Cerulean-ICDI_1.4 in HEK293 cells (grey). Overexpression of PKAc (red) reduces the binding.