XBART51582
PLoS One
January 1, 2015;
10
(11):
e0143363.
Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action.
Liang Q
,
Anderson WD
,
Jones ST
,
Souza CS
,
Hosoume JM
,
Treptow W
,
Covarrubias M
.
Abstract
Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the
nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltagegated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K
Shaw2 channel. Also, previous work suggested that the S4S5 linker of K
Shaw2 plays a role in the inhibition of this Kv channel by nalcohols and inhaled anesthetics. Here, we hypothesized that the S4S5 linker is also a determinant of the potentiation of
Kv1.2 and K
Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in
Kv1.2 (G329T) and K
Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally,
Kv1.2G329T impairs voltagedependent gating, which suggests that
Kv1.2 modulation by sevoflurane is tied to gating in a statedependent manner. Toward creating a minimal
Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of
Kv1.2 and
Kv1.2G329T by sevoflurane and other general anesthetics are T1independent. In contrast, the positive sevoflurane modulation of K
Shaw2 is T1dependent. In silico docking and molecular dynamicsbased freeenergy calculations suggest that sevoflurane occupies distinct sites near the S4S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.
PubMed ID:
26599217
PMC ID:
PMC4657974
Article link:
PLoS One
Grant support:
[+]
Genes referenced:
kcna2
kcnc2
Article Images:
[+] show captions

Fig 2. Analysis of GV relations from Kv1.2, ΔT1Kv1.2, KShaw2, KShaw2 T330G and ΔT1KShaw2.(A) Bestfit Boltzmann parameters (V1/2, z and Gmax) from individual paired measurements before (Ctr) and after exposure to 1 mM sevoflurane (Sevo). Each pair of symbols connected by a solid line represents an individual paired experiment (Materials and Methods). The Gmax graphs depict raw values before normalization (in mS). The P value resulting from a paired Studentt test is shown above each graph, and the red 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.


Fig 3. Modulation of the Kv1.2 channel by general anesthetics.(A) Effects of general anesthetics on the wholeoocyte Kv1.2 currents evoked by a voltage step to +60 mV from a holding voltage of 100 mV. Black, red and grey current traces correspond to control, anestheticexposed, and washout, respectively. The scale bars indicate 50 ms and 0.5 μA. (B) Concentrationresponse relations of various general anesthetics acting on the Kv1.2 channel. Solid lines are the best fits assuming the Hill equation for sevoflurane and nbutanol (Materials and Methods). Considering the magnitude of the change and the concentrations tested, only sevoflurane and nbutanol produced reliable Hill equation fits. N = 5–7 oocytes for each concentration. Bestfit parameters are summarized in Table 1.


Fig 4. Modulation of Kv1.2 FRAKT by general anesthetics.(A) Sequence alignment of the S4S5 linker from KShaw2 (314–332) and Kv1.2 (313–331) channels. Starting and ending residue numbers of the shown segments are indicated. In Kv1.2, the blue colored residues were swapped for the red colored residues in KShaw2 to create the Kv1.2 FRAKT mutant channel. (B) Effects of general anesthetics on wholeoocyte Kv1.2FRAKT currents evoked by a voltage step to +60 mV from a holding voltage of 100 mV. Black, red and grey current traces correspond to control, anestheticexposed, and washout, respectively. The scale bars indicate 50 ms and 0.5 μA. (C) Concentrationresponse relations of various general anesthetics acting on the Kv1.2 FRAKT channel. Solid lines are the best fits assuming the Hill equation (propofol and nbutanol) or a double Hill equation (sevoflurane, isoflurane, and halothane) (Materials and Methods). Due to the small magnitude of the chloroform results, no reliable Hill equation fit could be obtained. N = 5–8 oocytes for each concentration. Bestfit parameters are summarized in Table 1.


Fig 5. Kv1.2 G329T recapitulates the magnified positive modulation of Kv1.2 FRAKT by sevoflurane.(A) Effects of 1 mM sevoflurane on mutant wholeoocyte Kv1.2 currents evoked by a voltage step to +60 mV from a holding voltage of 100 mV. Black, red and grey current traces correspond to control, anestheticexposed, and washout, respectively. The scale bars indicate 50 ms and 1 μA. (B) Concentrationresponse relations of various general anesthetics acting on wild type and mutant Kv1.2 currents. Solid lines are the best fits assuming the double Hill equation (Materials and Methods). N = 4–8 oocytes for each dose. Bestfit parameters are summarized in Table 1.


Fig 6. Novel GV relations of Kv1.2 FRAKT and Kv1.2 G329T in the absence and presence of sevoflurane.(A) Families of wholeoocyte Kv1.2 FRAKT currents in the absence (left) and presence of 1 mM sevoflurane (right). 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 wholeoocyte Kv1.2 G329T currents in the absence (left) and presence of 1 mM sevoflurane (right). 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) GV relations of Kv1.2 FRAKT (red) and Kv1.2 G329T (blue) under control (open) or with 1 mM Sevoflurane (filled) (N = 6, 4, respectively). Solid lines are the best fits assuming a double Boltzmann equation (Materials and Methods). The bestfit parameters are summarized in Table A in S1 File.


Fig 7. Analysis of bimodal GV relations from Kv1.2 FRAKT, Kv1.2 G329T and ΔT1Kv1.2 FRAKT.(A) Bestfit double Boltzmann parameters (V1/2,1, z1, Gmax,1, V1/2,2, z2, Gmax,2) and Vmed from individual paired measurements before (Ctr) and after exposure to 1 mM sevoflurane (Sevo). Each pair of symbols connected by a solid line represents an individual paired experiment (Materials and Methods). The Gmax graphs depict raw values before normalization (in mS). The P value resulting from a paired Studentt test is shown above each graph, and the red marks indicate the mean values of the sample. (B)–(C) are as described for panel A. The number oocytes examined for each Kv channel was 6, 4, 5, respectively.


Fig 8. The T330G mutation eliminates the voltagedependent potentiation of the KShaw2 conductance by sevoflurane.(A) Families of wholeoocyte KShaw2 (N = 4) and KShaw2 T330G (N = 6) currents in the absence (left) and presence of 1 mM sevoflurane (right). 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) GV relations of KShaw2 (black) and KShaw2 T330G (red) in the absence (open) and presence of 1 mM sevoflurane (filled). Solid lines are the bestfits to the Boltzmann equation. Bestfit parameters are summarized in Fig 2 and Table A in S1 File. (C) The voltage dependence of the conductance ratio (GSevo/G0) KShaw2 (black) and KShaw2 T330G (red).


Fig 9. Positive modulation by sevoflurane is T1 domainindependent in Kv1.2 and Kv1.2FRAKT, and T1 domaindependent in KShaw2.(A) Concentrationresponse relations of various general anesthetics acting on ΔT1Kv1.2. Solid line is the best fit to the Hill equation for nbutanol. (B) Concentrationresponse relations of various general anesthetics acting on ΔT1Kv1.2 FRAKT. Solid lines are the best fits to the Hill equation (propofol and nbutanol) or double Hill equation (sevoflurane, isoflurane, and halothane). Bestfit parameters for results in panels A and B are summarized in Table 1. (C) Concentrationresponse relations of sevoflurane acting on KShaw2 and ΔT1KShaw2. Solid line is the bestfit double Hill equation to the KShaw2 data with the following parameters: K1 = 0.08 mM, A1 = 0.18, nH1 = 1, K2 = 4 mM, A2 = 1.4, nH2 = 1. These parameters are similar to those previously published for wild type KShaw2 (Table 1) [7]. KShaw2 and ΔT1KShaw2 were tested at +60 mV. N = 2–8 oocytes for each dose.


Fig 10. Putative sevoflurane binding sites in the Kv1.2 channel.(A) Representation of four distinct sevoflurane binding locations on Kv1.2: site 1 (light blue), 2 (blue), 3 (black) and 4 (purple). Each pair of subunits is represented in green and orange. Mutation G329T is highlighted yellow. (B) Closeup view of ligand binding sites and mutation. Note that site 2 is in close proximity to the mutated residue G329T. (C) Binding constants of individual sevoflurane sites. These estimates were obtained using the LIE method as described under S1 File. O and C stand for activationopen and restingclosed conformations of the channel.

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