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. 2017 Jan 6;6:e21989. doi: 10.7554/eLife.21989

Figure 5. Design schemes and experimental implementations of rapamycin-inducible CMI.

(A) Design principles for chemical-inducible CMI. Rapamycin simultaneously binds one FKBP and one FRB to combine any two FRB/FKBP-tagged components (PCRD and DCRD, in this version 1) selected from the three components of PCRD, DCRD and IQV. Thus, the three components would form the combinations (combination I, in the version 1) to satisfy the requirement of cooperative CMI (Figure 4E). (B) One representative implementation of chemical-inducible CMI. According to the design of version one in (A), PCRD-FRB and FKBP-DCRD were constructed and coexpressed with α1DS. Exemplars of Ca2+ current traces demonstrated that acute effects were induced by applying 1 μM rapamycin to the multi-component system of version 1 (lower panel), in contrast to α1DS control with no appreciable changes in ICa (upper panel). (C) Statistical summary of rapamycin-induced CMI (version 1). Averaged values over multiple cells (number indicated in parentheses) for the indices SCa (upper) or normalized Ipeak (lower) demonstrated time-dependent attenuations on both CDI and VGA upon rapamycin application as compared to the control group. (D) Rapamycin perfusion induced rapid translocation of YFP-FKBP-DCRD or YFP-FKBP-PCRD (version 2) onto the membrane by linking with FRB-CFP-Ras within 5 min, shown by confocal images via wide-field, CFP, and YFP channels. The local concentrations of YFP-FKBP-DCRD and YFP-FKBP-PCRD were substantially enhanced as suggested by the condensed YFP fluorescence at the membrane (outlined by CFP fluorescence). (E and F) According to rapamycin-inducible CMI of version 2, recombinant channels of α1DS-G12-FRB were coexpressed with cytosolic YFP-FKBP-PCRD and YFP-FKBP-DCRD. To enhance local concentrations of FKBP-tagged peptides near the channels, membrane-localized FRB-CFP-Ras was also overexpressed. Exemplars of Ca2+ current traces (E) exhibited strong attenuation (lower), in contrast to stable ICa from α1DS-G12-FRB alone (upper). In contrast to the control group, temporal profiles of rapamycin-inducible CMI (version 2) indicated strong attenuations on CDI (SCa, upper) and VGA (normalized Ipeak, lower). Based on totally five trials (cells), SCa changed from 0.77 ± 0.00 to 0.41 ± 0.06 and Ipeak was reduced to 35% ± 3% of the basal levels (F).

DOI: http://dx.doi.org/10.7554/eLife.21989.013

Figure 5.

Figure 5—figure supplement 1. Temporal profiles of fluorescent images for rapamycin-induced membrane-targeting.

Figure 5—figure supplement 1.

(A and B) Top rows: cartoon depicting the design of the rapamycin-induced translocation of DCRD (A) or PCRD (B) (Version 2 of chemical-inducible CMI). FRB-CFP-Ras was constitutively anchored to the plasma membrane by incorporating membrane-targeting segment from Ras protein. Lower rows: confocal images acquired via YFP channel showing rapid translocation of YFP-FKBP-DCRD (A) or YFP-FKBP-PCRD (B) to plasma membrane upon 1 μM rapamycin perfusion (left). At the time of ~120 s or later, YFP fluorescence was substantially condensed to overlap with the stable CFP fluorescence (FRB-CFP-Ras) on the membrane (right).
DOI: http://dx.doi.org/10.7554/eLife.21989.014
Figure 5—figure supplement 2. Detailed characterizations for rapamycin-induced CMI.

Figure 5—figure supplement 2.

(A and B) Voltage-dependent profiles of CDI and VGA exhibited no difference when α1DS channels were with (B) or without (A) rapamycin. Thick lines (red semitransparent) represent the CDI and VGA profiles from the α1DS control group. (C) Coexpression of CFP-PCRD-FRB and FKBP-DCRD-YFP with α1DS representing the control conditions before rapamycin did not cause any appreciable change in CDI and VGA, confirmed by pronounced SCa and JCa comparable to the values from α1DS control. (D) When rapamycin was applied to α1DS coexpressed with CFP-PCRD-FRB and FKBP-DCRD-YFP, SCa and JCa averages over different cells were attenuated with moderate potency (illustrated by green areas) presumably by rapamycin-induced linkage between PCRD and DCRD (version 1 of inducible CMI) to form the cooperation for effective CMI. (E) In rapamycin, with enhanced local concentrations of YFP-FKBP-PCRD and YFP-FKBP-DCRD (version 2 of rapamycin-induced CMI), the potency was much improved, demonstrated by more pronounced attenuations concurrently on both CDI and VGA (larger green areas in both r50 and Jpeak profiles).
DOI: http://dx.doi.org/10.7554/eLife.21989.015