Concentration-response parameters of the modulation of selected Kv channels by sevoflurane and other general anesthetics.

<p>* Sevoflurane tested at -20 mV, from Barber et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.ref007" target="_blank">7</a>].</p><p>Concentration-response parameters of the modulation of selected Kv channels by sevoflurane and other general anesthetics.</p

Novel <i>G</i>-<i>V</i> relations of Kv1.2 FRAKT and Kv1.2 G329T in the absence and presence of sevoflurane.

<p>(A) Families of whole-oocyte Kv1.2 FRAKT currents in the absence (<i>left</i>) and presence of 1 mM sevoflurane (<i>right</i>). Currents were evoked by step depolarizations from a holding voltage of -100 mV. The steps were delivered in increments of 10 mV from -90 to 130 mV. The scale bars indicate 100 ms and 2 μA. (B) Families of whole-oocyte Kv1.2 G329T currents in the absence (<i>left</i>) and presence of 1 mM sevoflurane (<i>right</i>). Currents were evoked by step depolarizations from a holding voltage of -100 mV. The steps were delivered in increments of 10 mV from -90 to 70 mV. The scale bars indicate 100 ms and 1 μA. (C) <i>G</i>-<i>V</i> relations of Kv1.2 FRAKT (<i>red</i>) and Kv1.2 G329T (<i>blue</i>) under control (<i>open</i>) or with 1 mM Sevoflurane (<i>filled</i>) (N = 6, 4, respectively). <i>Solid</i> lines are the best fits assuming a double Boltzmann equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). The best-fit parameters are summarized in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.s001" target="_blank">S1 File</a>.</p

Positive modulation by sevoflurane is T1 domain-independent in Kv1.2 and Kv1.2-FRAKT, and T1 domain-dependent in K-Shaw2.

<p>(A) Concentration-response relations of various general anesthetics acting on ΔT1-Kv1.2. <i>Solid</i> line is the best fit to the Hill equation for <i>n</i>-butanol. (B) Concentration-response relations of various general anesthetics acting on ΔT1-Kv1.2 FRAKT. <i>Solid</i> lines are the best fits to the Hill equation (propofol and <i>n</i>-butanol) or double Hill equation (sevoflurane, isoflurane, and halothane). Best-fit parameters for results in panels A and B are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.t001" target="_blank">Table 1</a>. (C) Concentration-response relations of sevoflurane acting on K-Shaw2 and ΔT1-K-Shaw2. <i>Solid</i> line is the best-fit double Hill equation to the K-Shaw2 data with the following parameters: <i>K</i>₁ = 0.08 mM, <i>A</i>₁ = 0.18, <i>n</i>_H1 = 1, <i>K</i>₂ = 4 mM, <i>A</i>₂ = 1.4, <i>n</i>_H2 = 1. These parameters are similar to those previously published for wild type K-Shaw2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.t001" target="_blank">Table 1</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.ref007" target="_blank">7</a>]. K-Shaw2 and ΔT1-K-Shaw2 were tested at +60 mV. N = 2–8 oocytes for each dose.</p

Modulation of the Kv1.2 channel by general anesthetics.

<p>(A) Effects of general anesthetics on the whole-oocyte Kv1.2 currents evoked by a voltage step to +60 mV from a holding voltage of -100 mV. <i>Black</i>, <i>red</i> and <i>grey</i> current traces correspond to control, anesthetic-exposed, and washout, respectively. The scale bars indicate 50 ms and 0.5 μA. (B) Concentration-response relations of various general anesthetics acting on the Kv1.2 channel. <i>Solid</i> lines are the best fits assuming the Hill equation for sevoflurane and <i>n</i>-butanol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). Considering the magnitude of the change and the concentrations tested, only sevoflurane and <i>n</i>-butanol produced reliable Hill equation fits. N = 5–7 oocytes for each concentration. Best-fit parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.t001" target="_blank">Table 1</a>.</p

Analysis of <i>G</i>-<i>V</i> relations from Kv1.2, ΔT1-Kv1.2, K-Shaw2, K-Shaw2 T330G and ΔT1-K-Shaw2.

<p>(A) Best-fit Boltzmann parameters (<i>V</i>_1/2, <i>z</i> and <i>G</i>_max) from individual paired measurements before (Ctr) and after exposure to 1 mM sevoflurane (Sevo). Each pair of symbols connected by a <i>solid</i> line represents an individual paired experiment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). The <i>G</i>_max graphs depict raw values before normalization (in mS). The <i>P</i> value resulting from a paired Student-<i>t</i> test is shown above each graph, and the <i>red</i> marks indicate the mean values of the sample. (B)–(E) are as described for panel A. The number oocytes examined for each Kv channel was 6, 6, 4, 6 and 6, respectively.</p

Kv1.2 G329T recapitulates the magnified positive modulation of Kv1.2 FRAKT by sevoflurane.

<p>(A) Effects of 1 mM sevoflurane on mutant whole-oocyte Kv1.2 currents evoked by a voltage step to +60 mV from a holding voltage of -100 mV. <i>Black</i>, <i>red</i> and <i>grey</i> current traces correspond to control, anesthetic-exposed, and washout, respectively. The scale bars indicate 50 ms and 1 μA. (B) Concentration-response relations of various general anesthetics acting on wild type and mutant Kv1.2 currents. <i>Solid</i> lines are the best fits assuming the double Hill equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). N = 4–8 oocytes for each dose. Best-fit parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.t001" target="_blank">Table 1</a>.</p

The T330G mutation eliminates the voltage-dependent potentiation of the K-Shaw2 conductance by sevoflurane.

<p>(A) Families of whole-oocyte K-Shaw2 (N = 4) and K-Shaw2 T330G (N = 6) currents in the absence (<i>left</i>) and presence of 1 mM sevoflurane (<i>right</i>). Currents were evoked by step depolarizations from a holding voltage of -100 mV. The steps were delivered in increments of 10 mV from -90 to +100 mV. The scale bars indicate 100 ms and 1 μA. (B) <i>G</i>-<i>V</i> relations of K-Shaw2 (<i>black</i>) and K-Shaw2 T330G (<i>red</i>) in the absence (<i>open</i>) and presence of 1 mM sevoflurane (<i>filled</i>). <i>Solid</i> lines are the best-fits to the Boltzmann equation. Best-fit parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.g002" target="_blank">Fig 2</a> and Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.s001" target="_blank">S1 File</a>. (C) The voltage dependence of the conductance ratio (<i>G</i>_Sevo/<i>G</i>₀) K-Shaw2 (<i>black</i>) and K-Shaw2 T330G (<i>red</i>).</p

Positive modulation of the Kv1.2 conductance by sevoflurane.

<p>(A) Families of whole-oocyte Kv1.2 currents before (<i>left</i>) and after exposure to 1 mM sevoflurane (<i>center</i>). Currents were evoked by step depolarizations from a holding voltage of -100 mV. The steps were delivered in increments of 10 mV from -50 to +50 mV. The overlay (<i>right</i>) directly compares selected currents in the absence (<i>black</i>) and presence (<i>red</i>) of sevoflurane (currents evoked by steps to the indicated voltages). (B) Normalized <i>G</i>-<i>V</i> relations of Kv1.2 in the absence (<i>black</i>) and presence of 1 mM sevoflurane (<i>red</i>) (N = 6). The solid lines are the best-fit Boltzmann functions. <i>G</i>_max is the control maximum conductance before exposure to sevoflurane (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). The mean best-fit parameters are summarized in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.s001" target="_blank">S1 File</a>. (C) The voltage dependence of the Kv1.2 conductance ratio (<i>G</i>_Sevo/<i>G</i>₀). This ratio was calculated from paired measurements of the <i>G</i>-<i>V</i> relations before (<i>G</i>₀) and after exposure to sevolfurane (<i>G</i>_Sevo).</p