Single channel kinetic analysis of the cAMP effect on I Ks mutants, S209F and S27D/S92D

Figures

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  Figure 1.. Subconductance analysis of Q1 S209F + E1 before and after 8-CPT-cAMP/Okadaic acid (OA). (a) Raw all-points histograms display the distribution of amplitudes from 20 active control sweeps (blue) and 20 sweeps in the presence of 200 µM 8-CPT-cAMP and 0.2 µM OA (red) from one representative cell were pulsed to + 60 mV for 4 s from a holding potential of −80 mV and filtered at 500 Hz. (b) Five initial thresholds used for idealization were 0.145, 0.22, 0.33, 0.5 and 0.75 pA and are shown in the table headers. The final idealization levels for control histograms were 0.13 ± 0.03, 0.23 ± 0.03, 0.35 ± 0.03, 0.50 ± 0.05 and 0.70 ± 0.10. In the presence of 8-CPT-cAMP/OA the levels were, 0.13 ± 0.03, 0.23 ± 0.03, 0.35 ± 0.03, 0.51 ± 0.05, 0.71 ± 0.07. (c) Total and mean dwell times (milliseconds) for each of the different thresholds, the percentage of time spent at each level, and the number of events at each threshold before and after 8-CPT-cAMP. These data were filtered at 0.5 kHz, and the bin width used was 0.01 pA. Only events longer than 1.5 ms were included. One-way ANOVA was used to compare control and 8-CPT-cAMP mean dwell times of each sublevel. The mean dwell times between control…

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  Figure 2.. Q1 S209F + E1 closed dwell times and burst analysis. (a) Closed dwell time distributions for S209F + E1 from 20 sweeps before and 20 sweeps after 200 µM 8-CPT-cAMP/0.2 µM OA from one representative cell. Data were fitted with the sum of two exponential functions. τ1: 1.11 ± 0.04 ms (area under the curve, AUC, 309 ± 5.5) and τ2: 8.61 ± 0.55 ms (AUC 75.4 ± 5.0 in control. After 8-CPT-cAMP/OA, τ1: 1.17 ± 0.06 ms (AUC 110 ± 3.9) and τ2: 8.66 ± 3.96 ms (AUC 14.2 ± 3.0). Bin width was 1 ms. (b) Probability distribution of closed time durations in control (black) and after 8-CPT-cAMP/OA (red), from data in A. (c) Probability distributions of burst durations based on open dwell time histograms, control (black) and after 8-CPT-cAMP/OA (red). Dwell-time histograms were fitted with the sum of three exponential functions in control τ1: 1.70 ± 0.09 ms (AUC 334 ± 10.7), τ2: 13.87 ± 2.41 ms (AUC 140 ± 16.7) and τ3: 44.38 ± 12.75 ms (AUC 39.5 ± 21.6). In 8-CPT-cAMP only the sum of two exponential functions could be fit, τ1: 2.62 ± 0.29 ms (AUC 88.2 ± 4.5) and τ2: 41.35 ± 3.36 ms (AUC 51.5 ± 2.7). Bin width was 2 ms. Only events longer than 1.5 ms in duration were used in this analysis.

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  Figure 3.. Subconductance analysis of Q1 S27D/S92D + E1 before and after 8-CPT-cAMP/OA. (a) Raw all-points histograms display the distribution of amplitudes from 20 active control sweeps (blue) and 20 sweeps in the presence of 200 µM 8-CPT-cAMP and 0.2 µM OA (red) (20 sweeps before and after 8-CPT-cAMP from one representative cell). (b) Five initial thresholds used for idealization were 0.145, 0.22, 0.33, 0.5 and 0.75 pA and are shown in the table headers. The final idealization levels for control histograms were 0.12 ± 0.03, 0.23 ± 0.03, 0.34 ± 0.04, 0.50 ± 0.05 and 0.71 ± 0.07. In the presence of 8-CPT-cAMP/OA the levels were, 0.11 ± 0.03, 0.23 ± 0.03, 0.34 ± 0.04, 0.51 ± 0.05, 0.74 ± 0.10. (c) Total and mean dwell times (milliseconds) for each of the different thresholds, the percentage of time spent at each level, and the number of events at each threshold before and after 8-CPT-cAMP. These data were filtered at 0.5 kHz, and the bin width used was 0.01 pA. Only events longer than 1.5 ms were included. A one-way ANOVA was used to compare control and 8-CPT-cAMP mean dwell times of each sublevel. None of the 8-CPT-cAMP mean dwell times were significantly different from control (p-value…

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  Figure 4.. Q1 S27D/S92D + E1 closed dwell times and burst analysis. (a).Closed dwell time distributions for S27D/S92D + E1 from 20 sweeps before and 20 sweeps after 200 µM 8-CPT-cAMP/0.2 µM OA (20 sweeps before and after 8-CPT-cAMP from one representative cell). Data were fitted with the sum of two exponential functions. τ1: 1.56 ± 0.06 ms (AUC 547 ± 20.3) and τ2: 7.18 ± 1.03 ms (AUC 158 ± 16.4) in control. After 8-CPT-cAMP/OA, τ1: 1.24 ± 0.14 ms (AUC 616 ± 132.3) and τ2: 3.44 ± 0.89 ms (AUC 416 ± 97.6). Bin width was 1 ms. (b) Probability distribution of closed time durations in control (black) and after 8-CPT-cAMP/OA (red), from data in A. (c) Probability distributions of burst durations based on open dwell time histograms, control (black) and after 8-CPT-cAMP/OA (red). Dwell-time histograms were fitted with the sum of three exponential functions in control τ1: 1.11 ± 0.12 ms (AUC 464 ± 56.8), τ2: 4.50 ± 0.88 ms (AUC 469 ± 56.9) and τ3: 11.10 ± 2.99 ms (AUC 126 ± 92.4). In 8-CPT-cAMP only the sum of two exponential functions could be fit, τ1: 1.59 ± 0.06 ms (AUC 792 ± 28.3) and τ2: 7.82 ± 1.52 ms (AUC 229 ± 20.1). Bin width was 2 ms. Only events longer than 1.5 ms in duration were used in…