Figure 4. Cooperation by PCRD, DCRD and IQ_V to induce CMI.

(A) Both CDI and VGA of α_1DS channels were attenuated by pre-linked PCRD-DCRD illustrated in the scheme (top), shown in the exemplar traces (middle) and voltage-dependent r₅₀ and J_peak profiles (lower two panels). The potency of CMI was indexed by S_Ca and J_Ca values and also illustrated by the green areas. Profiles of α_1DS control were indicated by thick semitransparent lines in red (lower two panels). (B) Both CDI and VGA of α_1DS-PCRD were strongly prohibited by DCRD. (C) Both CDI and VGA of α_1DS-G₂₄-DCRD were attenuated by PCRD. (D) FRET 2-hybrid assays demonstrated that pre-linked IQ_V-PCRD motif (YFP tagged) exhibited strong binding with CFP-DCRD, with higher binding affinity (K_d = 1135, units in donor-cube fluorescence intensity) than the binding affinity (K_d = 4700) between CFP-DCRD and YFP-IQ_V itself (without PCRD being fused). Additional PCRD peptides did not make any appreciable change (K_d = 4624) for the binding between CFP-DCRD and YFP-IQ_V, unable to rescue the low affinity back to the high level that the constitutive PCRD fusion (YFP-IQ_V-PCRD) could achieve. FR_max values for all these binding curves were similar (3.50—3.87). Each data point represented the FR (y-axis, FRET ratio) and D_free (x-axis, free donor concentration) values averaged over five adjacent individual cells sorted by D_free. (E) Working model to illustrate the collaboration among three components of PCRD, DCRD and IQ_V (embedded within α_1DS) for CMI induction. Grey circles represent the effective combinations, which require that at least two (out of three) components are closely engaged (e.g., fusion) to form the trio complex incorporating the third (separate) component. The three key combinations are I (IQ_V+PCRD-DCRD), II (IQ_V-PCRD+DCRD) and III (IQ_V-DCRD+PCRD), where ‘–’ denotes fusion or spatial closeness and ‘+’ indicates the separate peptide to be coexpressed. In addition, the positive control (IQ_V-PCRD-DCRD) also represents the native long variant α_1DL; and the three components (dotted circles in green, pink and blue) completely separate to each other (IQ_V+PCRD+DCRD) produce no CMI effect, serving as one negative control.

DOI: http://dx.doi.org/10.7554/eLife.21989.010

Figure 4—figure supplement 1. Functional similarities between PCRD_F and PCRD_D.

The pronounced CDI (S_Ca) and strong VGA (J_Ca) of α_1DS channels (indicated by red thick semitransparent lines in r₅₀ and J_peak plots) were similarly attenuated by PCRD-DCRD peptides made from either PCRD_F from Ca_V1.4 (left column) or PCRD_D from Ca_V1.3 (right column). Representative I_Ca traces (top), CDI and VGA profiles (panels in the two bottom rows) across the full voltage range were shown for the two types of peptides, with no significant difference. Green areas represent the potency of attenuation, supporting that PCRD_F and PCRD_D were interchangeable for CMI in this study as we assumed.

DOI: http://dx.doi.org/10.7554/eLife.21989.011

Figure 4—figure supplement 2. Cooperative perturbation of apoCaM/IQ_V binding by DCRD and PCRD.

(A) By 2-hybrid FRET with the CFP- or YFP-labeled constructs illustrated on top, IQ_V-PCRD (YFP-tagged) and CaM (CFP-tagged, apo-state) exhibited strong binding (blue), quantified by the maximum FRET ratio (FR_max) and effective equilibrium dissociation constant (K_d) from iterative curve fitting. The values of FR_max and K_d were indicated right above the fitted curves (in the same color) when applicable. Unfilled-dots and filled-dots represent individual cells and averaged results (over five cells) respectively. The binding between apoCaM and IQ_V-PCRD were severely perturbed by FKBP-DCRD (with FKBP tag adopted from subsequent rapamycin-inducible CMI, but without fluorescent tags of CFP/YFP), resulted into the data points (red) approaching the baseline of CFP/YFP mixture (cyan), indicative of very weak K_d. The perturbation was presumably by way of the close cooperation between FKBP-DCRD and IQ_V-PCRD to compete against apoCaM. The FRET binding analyses were consistent with the patch-clamp recordings confirming that CDI (S_Ca) of α_1DS-PCRD was attenuated in the presence of FKBP-DCRD (inset). (B) In the absence of PCRD, the perturbation by FKBP-DCRD was much less effective. Without the presence of PCRD, CFP-CaM and YFP-IQ_V (blue) were still able to bind. However, in contrast to the strong perturbation seen in (A), in the absence of PCRD, inclusion of DCRD peptides made no or very little difference in K_d and FR_maxvalues for CaM and IQ_V. The binding curve (dotted line in light blue) was replicated from CFP-CaM and YFP-IQ_V-PCRD (A) for comparison.

DOI: http://dx.doi.org/10.7554/eLife.21989.012