Amiodarone Inhibits Apamin-Sensitive Potassium Currents

<div><p>Background</p><p>Apamin sensitive potassium current (<i>I</i>_KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.</p><p>Objective</p><p>To test the hypothesis that amiodarone inhibits <i>I</i>_KAS in human embryonic kidney 293 (HEK-293) cells.</p><p>Methods</p><p>We used the patch-clamp technique to study <i>I</i>_KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.</p><p>Results</p><p>Amiodarone inhibited <i>I_KAS</i> in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of <i>I_KAS</i> induced with 1 µM intrapipette Ca²⁺ (n = 3). <i>I_KAS</i> inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of <i>I</i>_KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of <i>I</i>_KAS.</p><p>Conclusion</p><p>Both amiodarone and desethylamiodarone inhibit <i>I</i>_KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone's effects in preventing electrical storm in failing ventricles.</p></div

Inhibition of <i>I</i>_KAS with amiodarone is reversible.

<p>(A) Superimposed <i>I</i>_K traces in conditions as labeled. <i>I</i>_K was obtained with the ramp pulse protocol shown in the inset, and normalized with cell-capacitance. Intrapipette Ca²⁺ was 1 µM. (B) Time course of <i>I</i>_K measured at a membrane potential of 20 mV. The dotted line indicates the level of baseline <i>I</i>_K. AMD indicates amiodarone.</p

Inhibition of <i>I</i>_KAS with amiodarone is dose-dependent.

<p>(A) <i>I</i>_KAS was induced by the ramp pulse protocol used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070450#pone-0070450-g002" target="_blank">Figure 2</a>. (B) Time-course of <i>I</i>_K measured at 0 mV in the absence and presence of various amiodarone concentrations and apamin. The dotted line indicates the level of baseline <i>I</i>_K. (C) Dose-dependent inhibitory effects of amiodarone on <i>I</i>_KAS. The numbers in parenthesis indicate number of patches. The error bars represent S.E.</p

Desethylamiodarone also inhibits <i>I</i>_KAS.

<p>(A) Superimposed whole-cell <i>I</i>_K traces obtained by the same pulse protocol used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070450#pone-0070450-g001" target="_blank">Figure 1</a>. The intrapipette Ca²⁺ was 1 µM. (B) Current-voltage (I-V) relationships obtained in the absence and presence of 20 µM desethylamiodarone (DEA), and then 100 nM apamin. Similar results were observed in 6 cells.</p

Density of <i>I</i>_KAS and its inhibition by amiodarone is dependent on the intracellular Ca²⁺ concentration.

<p>(A) <i>I</i>_KAS densities obtained with three different intrapipette Ca²⁺ concentrations. The box-plot indicates the median and 25 and 75 percentile values. The whiskers indicate the minimum and maximum values. (B) Inhibitory effects of various concentrations of amiodarone on <i>I</i>_KAS induced with various intrapipette Ca²⁺ concentrations as shown in the top labels. In the left panel, inhibitory effects of 1 μM amiodarone were tested on <i>I</i>_KAS induced with two different intrapipette Ca²⁺ concentrations (1 µM and 500 nM). In the mid panel, inhibitory effects of 10 μM amiodarone were tested on <i>I</i>_KAS induced with two different intrapipette Ca²⁺ concentrations (1 µM and 500 nM). In the right panel, inhibitory effects of 30 μM amiodarone were tested on <i>I</i>_KAS induced with three different Ca²⁺ concentrations (1 µM, 500 nM and 250 nM). Inhibition of 250 nM Ca²⁺ induced <i>I</i>_KAS by 1 µM and 10 µM amiodarone is not shown since this was almost undetectable. The numbers in parentheses indicate the number of patches.</p

Inhibitory effect of amiodarone on <i>I</i>_KAS.

<p>(A) Representative superimposed whole-cell <i>I</i>_K traces obtained by the pulse protocol shown in the inset. The pipette solution contained 1 µM free Ca²⁺ to activate <i>I</i>_KAS. (B) Superimposed <i>I_K</i> traces in the presence of 10 µM amiodarone. (C) Superimposed <i>I</i>_K traces after adding 100 nM apamin to the same preparation. (D) Current-voltage (<i>I-V</i>) relationships obtained in the absence and presence of 10 µM amiodarone, then 100 nM apamin. <i>I</i>_K was measured between 280 ms and 290 ms of the test pulse, and plotted against membrane potentials. Similar results with 10 µM amiodarone were observed in 5 cells.</p

Inhibition of <i>I</i>_KAS with amiodarone is state-dependent.

<p>(A) Superimposed <i>I</i>_K traces in various conditions as labeled. The intrapipette Ca²⁺ was 500 nM. <i>I</i>_K was obtained with the same ramp pulse protocol used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070450#pone-0070450-g002" target="_blank">Figure 2</a> (also shown in the inset). (B) Superimposed <i>I</i>_K traces induced with 250 nM intrapipette Ca²⁺. Similar results were observed in 5 cells. AMD indicates amiodarone.</p

Additional file 1 of Gut microbiota dynamics and fecal SCFAs after colonoscopy: accelerating microbiome stabilization by Clostridium butyricum

Additional file 1: Figure S1. Faecal samples and intestinal contents were collected from 11 subjects at 8 time points before, during and 60 days after colonoscopy. NA denotes an incurred sample loss. Figure S2. The ratio of Firmicutes and Bacteroidetes showed the longitudinal fluctuation patterns of gut microbiota in the Control group. **p < 0.01. Figure S3. Quantity of buks containing bacteria stains at the phylum level. Figure S4. The ratio of Firmicutes and Bacteroidetes showed the longitudinal fluctuation patterns of gut microbiota in the Clostridium Butyricum group. **p < 0.01