Noceti F , Olcese R , Qin N , Zhou J , Stefani E .

AR-38970 NIAMS NIH HHS , GM17120-02 NIGMS NIH HHS , F32 GM017120 NIGMS NIH HHS

	Figure 2. Ca2+-dependent inactivation of Ca2+ current in the cloned cardiac Ca2+ channel α1Cβ2a. Superimposed traces evoked in an oocyte expressing α1C and β2a subunits, and the effect of DHP agonist Bay K 8644 (−). The voltage protocol was the same as in Fig. 1. (A) Voltage steps from HP −90 mV; Ba2+ was the charge carrier (10 mM Ba2+). (B) Currents recorded in 5 mM Ca2+. (C) Same voltage steps as in A, Ba2+ currents recorded in the presence of 50 nM Bay K 8644 (−). (D) Same voltage steps as in B, Ca2+ currents recorded in the presence of Bay K 8644 (−). Linear components were digitally subtracted by subpulses from the holding potential using the P/−4 method.
	Figure 3. Ca2+-dependent inactivation of Ca2+ currents in α1Cβ2a: effect of different Ca2+ concentrations. Superimposed traces evoked in an oocyte expressing α1Cβ2a. HP = −90 mV, SHP = −90 mV. (A) Voltage steps to −20, 0, +20 mV, in 2 mM Ca2+. The decay phase of the currents were fitted to double exponential functions. The rates of inactivation from the fits were: rf = 0.0114 ms−1, rs = 0.0031 ms−1 at −20 mV; rf = 0.0126 ms−1, rs = 0.0016 ms−1 at 0 mV; rf = 0.0065 ms−1, rs = not resolved at +20 mV. (B) Voltage steps to −10, +10, +30 mV in 5 mM Ca2+. The fitted rates were: rf = 0.0127 ms−1, rs = 0.0032 ms−1 at −10 mV; rf = 0.0173 ms−1, rs = 0.0027 ms−1 at +10 mV; rf = 0.0099 ms−1, rs = 0.0013 ms−1 at +30 mV. (C) Voltage steps to 0, −20, +40 mV in 10 mM Ca2+. The fitted rates were: rf = 0.0202 ms−1, rs = 0.0039 ms−1 at 0 mV, rf = 0.0224 ms−1, rs = 0.0035 ms−1 at +20 mV, rf = 0.0092 ms−1, rs = 0.0008 ms−1 at +40 mV.
	Figure 6. Summary plot of the voltage dependence of the fast and slow rates of Ca2+-dependent inactivation in α1Cβ2a: effect of Bay K 8644 (−). (A) Average values of the fast (empty symbols) and slow (filled symbols) rates of inactivation in 2 mM Ca2+ (○, fast, n = 11; •, slow, n = 13); 5 mM Ca2+ (□, fast, n = 13; ▪, slow, n = 14); 10 mM Ca2+ (▵, fast, n = 16; ▴, slow, n = 13). (B) Rates of Ca2+-dependent inactivation in 2 mM Ca2+ (⋄, fast, n = 10; ♦, slow, n = 8); 2 mM Ca2+ + 500 nM Bay K 8644 (−) (○, fast, n = 13; •, slow, n = 8); 5 mM Ca2+ + 500 nM Bay K 8644 (−) (□, fast, n = 15; ▪, slow, n = 11); 10 mM Ca2+ + 500 nM Bay K 8644 (−) (▵, fast, n = 12; ▴, slow, n = 11). All data are shown with their SEMs.
	Figure 4. Relative positions of the peak currents and the maximal inactivation on the voltage axis in α1C and α1Cβ2a. (A) Normalized rates and peak current values from the experiment in Fig. 3. Solid symbols are the normalized peak currents (I-V) and open symbols are the normalized rates (r-V) in 2 (○, •), 5 (□, ▪), and 10 (▵, ▴) mM Ca2+. The larger symbols on the normalized I-V represent the traces in Fig. 3 (shaded circles, squares, and triangles, respectively). The dotted lines show the voltage shift between I-V and r-V peaks. The maximal inactivation occurred at 0 (a), +10 (b), and +15 (c) mV in 10 mM Ca2+. The corresponding peaks of the I-Vs were: +10, +20, and +25 mV, respectively. (B) Normalized rates and peak current values in α1C alone in 5 mM Ca2+. The points are mean values ± SEM (n = 7 for the I-V and n = 8 for the r-V). The dotted line shows the voltage shift between I-V and r-V peaks.
	Figure 5. Ca2+-dependent inactivation and effect of Bay K 8644 (−) at three different Ca2+ concentrations in α1Cβ2a. Superimposed traces evoked in an oocyte expressing α1Cβ2a. HP = −90 mV, SHP = −90 mV. (A) Voltage steps to −20, 0, +20 mV in 2 mM Ca2+. The decay phase of the currents were fitted to double exponential functions. The rates of inactivation from the fits were: rf = 0.0127 ms−1, rs = 0.0032 ms−1 at −20 mV; rf = 0.0164 ms−1, rs = 0.0021 ms−1 at 0 mV; rf = 0.0116 ms−1, rs = 0.0016 at +20 mV. (B) Voltage steps to −25, −5, +15 mV in 2 mM Ca2+ and 500 nM Bay K 8644 (−). The fitted rates were: rf = 0.0333 ms−1, rs = 0.0056 ms−1 at −25 mV; rf = 0.0336 ms−1, rs = 0.0045 ms−1 at −5 mV; rf = 0.0132 ms−1, rs = 0.0016 ms−1 at +15 mV. (C) Voltage steps to −15, +5, +25 mV in 5 mM Ca2+ and 500 nM Bay K 8644 (−). The fitted rates were: rf = 0.0398 ms−1, rs = 0.0063 ms−1 at −15 mV, rf = 0.0395 ms−1, rs = 0.0044 ms−1 at +5 mV, rf = 0.0150 ms−1, rs = 0.0020 ms−1 at +25 mV. (D) Voltage steps to −5, +15, +35 mV, in 10 mM Ca2+ and 500 nM Bay K 8644 (−). The fitted rates were: rf = 0.0466 ms−1, rs = 0.0057 ms−1 at −5 mV, rf = 0.0399 ms−1, rs = 0.0045 ms−1 at +15 mV, rf = 0.0148 ms−1, rs = 0.0023 ms−1 at +35 mV.
	Figure 7. Relative positions of the peak currents and the maximal inactivation on the voltage axis in α1Cβ2a in the presence of Bay K 8644 (−). Normalized rates and peak current values in α1Cβ2a in 5 mM Ca2+ and in the presence of 500 nM Bay K 8644 (−). The points are mean values ± SEM (n = 4 for I-V and n = 3 for r-V). The dotted line shows the voltage shift between I-V and r-V peaks.
	Figure 8. Ca2+-dependent inactivation and effect of Bay K 8644 (−) at three different Ca2+ concentrations in α1C. Superimposed traces evoked in an oocyte expressing α1C. HP = −90 mV, SHP = −90 mV. (A) Voltage steps to −10, +10, +30 mV in 2 mM Ca2+. The decay phase of the currents was fitted to a single exponential function. The rates of inactivation from the fits were: r = 0.0027 ms−1 at −10 mV, r = 0.0028 ms−1 at +10 mV, r = 0.0021 at +30 mV. (B) Voltage steps to −15, +5, +25 mV in 2 mM Ca2+ and 500 nM Bay K 8644 (−). The decay phase of the currents were fitted to double exponential functions. The fitted rates were: rf = 0.0192 ms−1, rs = 0.0016 ms−1 at −15 mV; rf = 0.0277 ms−1, rs = 0.0038 ms−1 at +5 mV; rf = 0.0073 ms−1, rs = 0.0007 ms−1 at +25 mV. (C) Voltage steps to −10, +10, +30 mV in 5 mM Ca2+ and 500 nM Bay K 8644 (−). The fitted rates were: rf = 0.0255 ms−1, rs = 0.0038 ms−1 at −10 mV, rf = 0.0356 ms−1, rs = 0.0055 ms−1 at +10 mV, rf = 0.0079 ms−1, rs = 0.0012 ms−1 at +30 mV. (D) Voltage steps to −5, +15, +35 mV in 10 mM Ca2+ and 500 nM Bay K 8644 (−). The fitted rates were: rf = 0.0299 ms−1, rs = 0.0041 ms−1 at −5 mV, rf = 0.0418 ms−1, rs = 0.0053 ms−1 at +15 mV, rf = 0.0159 ms−1, rs = 0.0035 ms−1 at +35 mV.
	Figure 9. Time course of the effect of BAPTA: the removal of Ca2+-dependent inactivation. (left) BAPTA-perfused oocyte; (right) control oocyte. (A) Currents elicited by pulsing to 10 mV from an HP of −90 mV in an oocyte injected with α1Cβ2a (SHP = −90 mV) in the presence of 5 mM Ca2+. The oocyte was perfused with BAPTA 500 mM at the speed of 1 ml/h. a was acquired at time t = 1 min 15 s; b at t = 1 min 46 s; c at t = 4 min 26 s; d at t = 9 min 48 s; e at t = 14 min 29 s; f at t = 20 min 05 s. The initial phase of decay (first 250 ms) in c–f was fitted to a single exponential. The rates of inactivation were: 0.0099 ms−1 (c), 0.0077 ms−1 (d), 0.0039 ms−1 (e), and 0.0025 ms−1 (f). (B) Time course of the removal of Ca2+-dependent inactivation. Traces in A are indicated with the corresponding letters. (C) Time course of peak current at 0 mV in 5 mM Ca2+ in a control nonperfused oocyte injected with 100 nl of BAPTA 50 mM (see materials and methods). The control current shows rundown over the time course of the experiment. (D) Lack of change in the voltage dependence of the fast rate of the Ca2+-dependent inactivation measured at the beginning (t = 0, •) and at the end of the control experiment (t = 23 s, ○). (E) Fast (▵) and slow (▴) rates of Ca2+-dependent inactivation for the current plotted in C, during the time course of the control experiment.
	Figure 10. Model of Ca2+-dependent inactivation. Three modes of L-type Ca2+ channels are originating from a single deep closed state (C01). The top line has no opening: the channel remains silent. The middle and bottom lines end with open states and subsequent Ca2+-dependent inactivated states. The rates (α, β), charges (z), and fractions of the field (δ) that have been used for the fits and simulation are shown in Table I, and they refer to the kinetic transitions as follows: α01, β01, z1, δ1: C01 ↔ C02; α02, β02, z2, δ2: C02 ↔ C03; α03, β03, z3, δ3: C03 ↔ O04; α04, β04: O04 ↔ I05, Ca2+-dependent, voltage-independent transition; α12, β12, z2, δ2: C12 ↔ C13; α13, β13, z3, δ3: C13 ↔ C14; α22, β22, z2, δ2: C22 ↔ C23; α23, β23, z3, δ3: C23 ↔ O24; α24, β24: O24 ↔ I25, Ca2+-dependent, voltage-independent transition; αn, βn: C02 ↔ C12, voltage-independent transition; αbk, βbk: C02 ↔ C22, voltage-independent transition; αb, βb: C03 ↔ C23, voltage-independent transition.
	Figure 11. Fits and simulations of α1Cβ2a Ca2+ currents. (A) Current traces recorded from an oocyte expressing α1Cβ2a, in response to voltage stimuli of −20, −10, 0, +10, and +20 mV. The traces are superimposed with their fits, generated by the model-fitting procedure described in materials and methods. (B) I-V and r-V are from the simulated traces in A. The current peaks at 10 mV, while the r-V peaks at 0 mV. The values for the fast rates of inactivation were obtained by fitting the decay phase of the simulated current with a double exponential function.

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