Figure 6. Mechanistic insights enlightened by unique features of CMI.

(A) Temporal profiles between CMI and conventional inhibitions were compared. For rapamycin-inducible CMI (two versions in the top two rows of panels), representative Ca²⁺ current traces in rapamycin were selected from sequential time-points (left column) and superimposed together (middle column) for comparison. The time sequence was indicated by color: the first in pink, intermediate in grey, and the last in blue. I_peak exhibited the trend of inhibition but not for I₃₀₀ (I_Ca at 300 ms) (right column). In contrast, for run-down process and isradipine blockage (bottom two rows), both I_peak and I₃₀₀ exhibited the declining trends indicating substantial inhibitions. (B) Ca²⁺ current density at 300 ms (J_300,Ca) remained at the same level (indicated by the J₃₀₀ values at −10 mV), for various channel variants under test, regardless of whether CMI was in effect. Thick lines (semitransparent in red) represent the J_300,Ca profile of α_1DS control. (C) Functional and the structural insights into the two modes of inhibition. DCT/apoCaM-dependent CMI and Ca²⁺/CaM-mediated CDI result in indistinguishable gating (green arrows) appearing due to similar causes, i.e., either total or partial loss of the apo-state CaM/IQ_V complex. That says, in CMI, apoCaM pre-association is totally lost; and in CDI, apoCaM is calcified and dislocated from the pre-association sites. Although the triggers are different for CMI vs CDI, i.e., DCT competing (off apoCaM) vs Ca²⁺ binding (onto apoCaM), the similarities between the two different inhibitory regulations of CMI and CDI invite the hypothesis that the core gating machinery (cyan squares) upon depolarization might step into the same scenario/mode including structural details (red arrows). A series of current traces (on the right) indicate CMI with different potency (enhanced from pink to cyan, upper), in comparison with the trace at different stages of CDI (developed from pink to cyan, lower) superimposed with the trace from ultrastrong CMI (dotted trace in cyan). These analyses point to one important notion that the lower limit of CMI is determined by end-stage CDI (the traces or the phases in cyan), and thus the dynamic changes for CMI effects on α_1DS (indexed with I_peak and S_Ca) are preset, i.e., from the pink (no CMI and ultrastrong CDI) to the cyan (ultrastrong CMI and no CDI).

DOI: http://dx.doi.org/10.7554/eLife.21989.016

Figure 6—figure supplement 1. Detailed comparisons among CMI, run-down and blockage.

CMI was compared with conventional inhibitions/inhibitors including channel blockage (by isradipine) and run-down process (reduction of channel numbers), indexed with S_Ca, I_peak and I₃₀₀. Both rapamycin-induced CMI and run-down/blockage exhibited time-dependent decreases in I_peak, as expected from effective I_Ca inhibitions (B). In contrast, CDI (S_Ca) did not change in run-down/blockage; whereas for CMI S_Ca exhibited rapamycin-dependent attenuation, similar to I_peak (A). Moreover, the plateau of I_Ca (I₃₀₀) remained constant in spite of I_peak attenuation in CMI, whereas I₃₀₀ changed (decreased or recovered) in a similar time-course to that of I_peak during run-down and blockage (C). Since S_Ca is in proportion to I_peak/I₃₀₀, the two major types of inhibitions (CMI vs conventional inhibitions) were distinct within this context: for CMI, both I_peak and S_Ca changed but with I₃₀₀ being kept constant; in contrast, for conventional inhibitions, both I_peak and I₃₀₀ changed, but S_Ca remained unaltered.

DOI: http://dx.doi.org/10.7554/eLife.21989.017

Figure 6—figure supplement 2. Indices at different time points of I_Ca to quantify CMI and end-stage CDI.

(A) Analyses at both 300 ms and 1000 ms were able to reliably unveil that channels subject to CMI were essentially tuned to approach I_Ca levels in end-stage CDI. Upper panel, we set I₁₀₀₀ (I_Ca at 1000 ms) of both exemplar traces to the same level for α_1DS and α_1DS-PCRD-DCRD (in principle, they should be the same); and the levels of I_Ca at 300 ms (I₃₀₀) turned out to be nearly the same, similarly as the analyses with I₁₀₀₀. At 300 ms, the kinetic difference between I_Ba (normalized to the peak of I_Ca) and I_Ca was very little, indicative of end-stage CDI (upper right). Full profiles of Ca²⁺ current density (indexed with J₃₀₀ or J₁₀₀₀) were indistinguishable for both groups of α_1DS and α_1DS-PCRD-DCRD, further supporting that both are qualified as reliable indices for CMI analysis. (B) For rapamycin-inducible CMI (version 2) by supplying PCRD, DCRD and α_1DS with membrane-targeting and rapamycin-inducible mechanisms, representative I_Ca traces were selected from sequential time-points (left), and the first (purple) and the last (cyan) traces were superimposed together for comparison (right). I_peak exhibited a trend of time-dependent decrease; meanwhile, both I₃₀₀ and I₁₀₀₀ remained rather constant (dotted outlines in black, blue and pink respectively). (C) Statistical summary of I_Ca amplitudes for rapamycin-induced CMI (version 2). I_Ca values were measured at different time points, i.e., I_peak, I₁₀₀, I₃₀₀ and I₁₀₀₀, each of which was either rescaled to I_peak (left) or to its own (right). I_peak exhibited robust inhibition due to CMI, and a trend of moderate decline (right) was noticeable from I₁₀₀ (I_Ca at 100 ms); whereas I₃₀₀ and I₁₀₀₀ clearly remained constant throughout the full time-course, thus reliably representing the end stage or steady state of CDI. In line with our previous analyses, the current level of I₃₀₀ or I₁₀₀₀ determined the lower limit of I_Ca (measured by I_peak) subject to CMI of the maximum potency.

DOI: http://dx.doi.org/10.7554/eLife.21989.018

Figure 6—figure supplement 3. The high-affinity binding between DCRD and the channel was not perturbed by Ca²⁺/CaM.

(A) Upon applying ionomycin, an ionophore massively raising intracellular Ca²⁺ levels, endogenous CaM should switch from Ca²⁺-free state (apoCaM) to Ca²⁺-bound state (Ca²⁺/CaM). However, similar K_d values were obtained by fitting the FRET binding curves before and after ionomycin application (pink, before ionomycin; red, in ionomycin), indicating that Ca²⁺/CaM was unable to perturb the strong binding between DCRD and IQ_V-PCRD. In the FR-D_free plots, unfilled-dots and filled-dots represent individual cells and averaged results (over five cells) respectively. (B) Following ionomycin administration, the indices (K_d and FR_max) of the binding between CaM and IQ_V were clearly strengthened consistent with the IQ_V/CaM interactions previously established.

DOI: http://dx.doi.org/10.7554/eLife.21989.019