Kinetics insight into the roles of the N- and C-lobes of calmodulin in RyR1 channel regulation

Figures

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  Figure 1. Steady-state fluorescence measurement ofAcrCaM,AcrCaMC, andAcrCaMNin a solution of 20 mM Mops, 30 mM NaCl (pH 7), containing 0.01 to 100 μM [Ca2+]. Each panel includes cartoon schematics describing the acrylodan-labeled CaM (AcrCaM) and its lobe-specific Ca2+-sensitive mutants retaining Ca2+ binding sites at N-lobe (AcrCaMN) or C-lobe (AcrCaMC), respectively. The fluorophore (green star) was attached at positions T34C or T110C as Acr34CaMx and Acr110CaMx accordingly. The orange circles illustrate the Ca2+ binding sites at each lobe, crossed orange circles indicate ablated Ca2+ binding sites. A, fluorescence responses to [Ca2+] of Acr34CaM, Acr34CaMC, and Acr34CaMN. B, fluorescence response to [Ca2+] of Acr110CaM, Acr110CaMC, and Acr110CaMN. The blue traces represent the shift in peak-wavelength of the fluorescence spectrum in response to [Ca2+]. The black traces represent the change in peak-intensity of the fluorescence spectrum in response to [Ca2+]. Solid lines represent fits using the Hill function. KD values and Hill coefficients are means ± SD of fits of the experimental data to Hill equations (n = 3). Acr, acrylodan; CaM, calmodulin.

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  Figure 2. Transient kinetics of Ca2+binding to free CaM and its lobe-specific Ca2+sensitive mutants CaMNand CaMC. Ca2+ dissociation (blue curves) and association (red curves) with acrylodan attached at residue 34 (left column) or 110 (right column), respectively. Blue curves: CaM (0.5 μM) presaturated with 30 μM Ca2+ was rapidly mixed with an equal volume of 2 mM EGTA in a stopped-flow apparatus, and the fluorescence change was monitored at 4 °C. Red curves: CaM (0.5 μM) was rapidly mixed with an equal volume of 20 μM free Ca2+ (EGTA buffered) at 4 °C in 20 mM Mops, 30 mM NaCl, pH 7. The data were fitted with one- or two-exponential functions using Pro-Data Software Suite (version 4.2.12, Applied Photophysics) (dotted lines), and fitting results were summarized in Table 1. CaM, calmodulin.

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  Figure 3. Kinetics of Ca2+ binding to the CaM/RyR1 complex.A, Ca2+ dissociation from Acr34CaM in the absence (dotted curves) and presence of excess purified RyR1 (solid curves). B, Ca2+ association to Acr34CaM in the absence (dotted curves) and presence of excess purified RyR1 (solid curves). The experiments were performed as described in Figure 2, except for the 2 h incubation of CaM with RyR1, in a solution containing 20 mM Pipes, 150 mM KCl, 5 mM GSH, 1 μg/ml of aprotinin/leupeptin, and 2 mM DTT, pH 7. The final concentration of RyR1-CaM binding sites was ∼2.5-fold of the added [CaM]. Fitting results are shown as means ± SD (n = 3) and summarized in the corresponding inset tables. Acr, acrylodan; CaM, calmodulin; RyR, ryanodine receptor.

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  Figure 4. Kinetics of Ca2+binding to CaMCand CaMNin complex with RyR1. Ca2+ dissociation/association to Acr34CaMC (A and B) and Acr34CaMN (C and D) in the absence (dotted curves) and presence (solid curves) of purified RyR1. Experimental conditions and data analysis are the same as in Figure 3. Fitting results are shown as means ± SD (n = 3) and summarized in the corresponding inset tables. Acr, acrylodan; CaM, calmodulin; RyR, ryanodine receptor.

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  Figure 5. Ca2+binding to oximimetic CaM mutant M124Q free in solution.A, steady-state fluorescence of Acr34M124Q. Top panel: fluorescence peak-wavelength versus [Ca2+]. Bottom panel: peak-intensity versus [Ca2+], as described in Figure 1. Open squares represent Acr34CaM, and closed squares represent Acr34M124Q. Solid lines are fits to the Hill equation. B, time courses of Ca2+ binding events of Acr34M124Q (solid traces) and Acr34CaM (dotted traces) under the same conditions as described in Experimental procedures. C, Ca2+ affinity (KD) and rate constants of Ca2+ association/dissociation from Acr34CaM and Acr34M124Q obtained from (A) and (B), respectively. KD values are fits of the experiments to the Hill equation, mean ± SD (n = 3). Rate constants are shown as means ± SD (n ≥ 3). Acr, acrylodan; CaM, calmodulin.

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  Figure 6. Kinetics of Ca2+binding to oximimetic CaM mutant M124Q in complex with RyR1. Kinetics of Ca2+ dissociation (A) and association (B) to Acr34M124Q in the presence of purified RyR1. The dotted curves represent the corresponding controls in the absence of RyR1. The experimental conditions and data analysis are the same as in Figure 3. Rate constants are shown as means ± SD (n = 3) and summarized in the corresponding inset tables. Acr, acrylodan; RyR, ryanodine receptor.

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  Figure 7. Model of Ca2+ binding to CaM free and in complex with RyR1.A, Ca2+ association to the C-lobe of free CaM is faster than to the N-lobe, and Ca2+ dissociation from the N-lobe CaM is faster than from the C-lobe. Apo-CaM is more compact (left) than Ca2+-bound CaM (center and right) (19). B, RyR1 binding to CaM does not alter the overall sequence of Ca2+ binding to the two lobes of CaM. Fast Ca2+ binding to the C-lobe initiates the inhibition of RyR1 at high [Ca2+] while the slow Ca2+ association to the N-lobe reinforces the timely inhibition. Upon Ca2+ return to the basal level, fast Ca2+ dissociation from the N-lobe enables the return of CaM to its state of RyR1 activator at low [Ca2+]. Two Ca2+ binding sites in each domain are indicated in the apo- or calcium-loaded state. The illustrated locations of CaM on RyR are based on cryo-EM structures for apo-CaM/RyR1 (left) (15), Ca2+-CaM/RyR1 (center) (17), whereas the location of Ca2+-CaM on the inhibited RyR1 (right) reflects FRET-based trilateration results (20) and the Ca2+-CaM/RyR2 structure (14). “Fast” and “slow” indicate the rate constants of Ca2+-association and dissociation from the CaM. Ca2+ association and dissociation from…