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Figure 1. Gating currents from oocytes expressing the truncated and full-length forms of the rabbit skeletal muscle L-type Ca2+ channel α1 subunit (α1SΔC and α1S). (A) Voltage-clamp traces recorded in six oocytes during 20-ms depolarizing steps to the values shown on the left from a holding potential of −90 mV. The combination of cRNAs injected is shown above each set of traces. Each trace is the average of four responses filtered at 1 kHz and sampled at 20 kHz. The dotted lines represent zero current. (B) Qon vs. −Qoff plot for the α1SΔC, β1b-injected oocyte from A, middle right, shows charge conservation. A straight line with a slope of 1 is shown as a reference. Values of Qon and Qoff were obtained by integrating the gating currents during and after the pulse. 10 datum points (0.5 ms) near the end of the pulse were used to define the baseline for Qon, while 10 datum points near the end of the trace (3–5 ms after the end of the pulse) were used to define the baseline for Qoff. Qon was integrated throughout the pulse to allow slow components of charge movement to be included. Qoff did not have prominent slow components and was integrated over the first ∼3 ms after the pulse. A number of cells showed small, slow tail currents at depolarized potentials (see +20 mV traces in A, top right and middle left) that were excluded from Qoff by baseline subtraction. (C) Qon (•) and Qoff (▾) versus voltage for the same oocyte were fitted by Boltzmann distributions of the form: Q-V = Qmax/{1 + exp[−(V − V1/2Q)/kQ]}, where Qmax is the maximal charge moved at depolarized voltages, V1/2Q is the voltage at which charge movement is half maximal, and kQ describes the steepness of the distribution. The solid line shows the average of the Qon and Qoff fits: Qmax = 175 pC, V1/2Q = −25.9 mV, and kQ = 15.6 mV.
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Figure 2. The magnitude and voltage dependence of charge movement in oocytes expressing α1SΔC and α1S. (A) Qoff versus voltage from five oocytes expressing α1S, β1a, α2δ, and γ (□) and 12 oocytes expressing α1SΔC, β1a, α2δ, and γ (▪). The solid and dashed lines through the data points show the mean Boltzmann fits to the data from individual cells (fitted as described in Fig. 1). α1S/β1a/α2δ/γ: Qoff, max = −82.9 ± 32.8 pC, V1/2Q = −24.7 ± 2.2 mV, kQ = 13.1 ± 1.0 mV, n = 5; α1SΔC/β1a/α2δ/γ: Qoff, max = −175 ± 34.0 pC, V1/2Q = −22.2 ± 0.8 mV, kQ = 16.1 ± 0.3 mV, n = 12. The differences in Qoff, max between these two data sets are not statistically significant (P = 0.13; see text). (B) Qoff versus voltage from nine oocytes expressing α1S, β1b, α2δ, and γ (○), eight oocytes expressing α1SΔC, β1b, α2δ, and γ (•), six oocytes expressing the auxiliary subunits alone (♦), and eight uninjected oocytes (▪). Error bars correspond to the standard error of the mean and are smaller than the symbol when not visible. Gating currents were recorded in 2 mM extracellular Co2+, and Qoff was calculated as described in Fig. 1. The solid and dashed lines through the symbols are mean Boltzmann fits, as above. α1S/β1b/α2δ/γ: Qoff, max = −69.0 ± 11.0 pC, V1/2Q = −23.9 ± 1.3 mV, kQ = 16.2 ± 0.5 mV, n = 9; α1SΔC/β1b/α2δ/γ: Qoff, max = −137 ± 20.1 pC, V1/2Q = −27.5 ± 3.2 mV, kQ = 15.9 ± 1.4 mV, n = 8. The differences in Qoff, max between these two data sets are statistically significant (P = 0.008; see text). (C) Normalized Qoff versus voltage (Q-V) curves for oocytes expressing α1S (open symbols) and α1SΔC (filled symbols) with β1a, α2δ, and γ (squares) or β1b, α2δ, and γ (circles). The Q-V curves are compared with the mean G-V curve measured in 10 mM Ba2+ solution for oocytes expressing α1SΔC, β1b, α2δ, and γ (▾). To obtain the Q-V curves, data from individual oocytes were fitted by Boltzmann distributions as described above and normalized by Qoff, max. To obtain the G-V curve, inward L-type currents were evoked by 300-ms depolarizations in 10 mM extracellular Ba2+, filtered at 1 kHz, sampled at 2 kHz, and measured at the end of the pulse. For eight individual oocytes, the I-V curves so obtained were fit to the function: I-V = Gmax(V − Erev)/{1 + exp[−(V − V1/2G)/kG]}, where Gmax is the maximal conductance attained, Erev is the current reversal potential, V1/2G is the half-activation potential, and kG describes the steepness of activation. In the fitting procedure, Gmax, Erev, V1/2G, and kG were all allowed to be free parameters. Normalized G-V curves were then obtained by dividing each I-V curve by Gmax(V − Erev). The lines in C show the average normalized Boltzmann distributions fit to the Q-V and G-V relations measured in individual cells. The fitted parameters for the Q-V curves are given for A and B, above. For the G-V curve: Gmax = 3.4 ± 0.3 μS, V1/2G = 5.9 ± 0.6 mV, and kG = 9.4 ± 0.2 mV.
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Figure 3. Ionic and gating currents recorded in the same cell for oocytes expressing α1S plus auxiliary subunits, α1SΔC plus auxiliary subunits, or auxiliary subunits alone. For each oocyte, inward ionic currents were recorded in 10 mM Ba2+ solution. The Ba2+ solution was replaced by 2 mM Co2+ solution (applied in the bath using a Pasteur pipet), and then gating currents were recorded after capacitance compensation. In A–E, the ionic currents evoked by test pulses to −40 and +20 mV from the holding potential of −90 mV are shown on a slow time scale (top, average of two traces in each case), and the gating currents evoked by test pulses to the same voltages are shown on a much faster time scale (bottom, average of four traces). The lighter traces show the responses at −40 mV, and the darker traces show the responses at +20 mV. Ionic currents were evoked by 300-ms depolarizations, filtered at 1 kHz and sampled at 2 kHz, and are presented with the residual capacitance transients blanked. Gating currents were evoked by 20-ms depolarizations, filtered at 1 kHz and sampled at 20 kHz.
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Figure 4. Comparison of ionic current to gating charge movement in oocytes expressing α1S or α1SΔC plus the auxiliary subunits. (A–D) Qon versus voltage (left scale) and current versus voltage (right scale) measured in the same oocytes. (A) Data from six cells expressing α1S, β1a, α2δ, and γ. (B) Data from five cells expressing α1SΔC, β1a, α2δ, and γ. (C) Data from 10 cells expressing α1S, β1b, α2δ, and γ. (D) Data from nine cells expressing α1SΔC, β1b, α2δ, and γ. Experiments were performed as in Fig. 3. Qon, measured as described in Fig. 1, was used instead of Qoff in these experiments to avoid the possibility of contamination with residual Ba2+ tail current. The solid lines are the mean Boltzmann Q-V curves determined as in Fig. 2 for each set of data. In A, Qon, max = 140 ± 21.6 pC, V1/2Q = −12.3 ± 5.5 mV, kQ = 16.0 ± 1.7 mV, Imax = −8.5 ± 1.1 nA, and n = 6. In B, Qon, max = 165 ± 47.6 pC, V1/2Q = −9.2 ± 3.3 mV, kQ = 15.6 ± 2.3 mV, Imax = −53.0 ± 8.6 nA, and n = 5. The difference in Qon, max between the data in A and in B was not statistically significant (P = 0.63). In C, Qon, max = 80.1 ± 9.6 pC, V1/2Q = −24.6 ± 1.8 mV, kQ = 13.8 ± 1.4 mV, Imax = −7.3 ± 1.1 nA, and n = 10. In D, Qon, max = 125 ± 16.0 pC, V1/2Q = −27.4 ± 2.6 mV, kQ = 14.4 ± 1.8 mV, Imax = −68.8 ± 7.2 nA, and n = 6. The difference in Qon, max between C and D was statistically significant (P = 0.02). (E) Ratio of maximum Ba2+ current (Imax) to Qon, max in cells expressing α1SΔC or α1S plus auxiliary subunits. For each cell, the maximum inward current evoked by a 300-ms depolarization to +10 or +15 mV in 10-mM Ba2+ solution was divided by the value of Qon, max determined from the Boltzmann fit to the Qon versus voltage relationship. In cells expressing the β1a subunit, Imax/Qon, max was 0.06 ± 0.01 nA/pC when α1S was expressed and 0.37 ± 0.05 nA/pC when α1SΔC was expressed, a difference which was statistically significant (P = 4.8 × 10−6). In cells expressing the β1b subunit, Imax/Qon, max was 0.10 ± 0.01 nA/pC when α1S was expressed and 0.60 ± 0.09 nA/pC when α1SΔC was expressed, a difference which was also statistically significant (P = 6.2 × 10−5).
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