Liang Q et al. (2015), Positive Allosteric Modulation of Kv Channels b...

XB-ART-51582

PLoS One 2015 Nov 24;1011:e0143363. doi: 10.1371/journal.pone.0143363.

Show Gene links Show Anatomy links

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 .

???displayArticle.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 voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 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, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent 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 Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 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.

???displayArticle.pubmedLink??? 26599217
???displayArticle.pmcLink??? PMC4657974
???displayArticle.link??? PLoS One
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: kcna2 kcnc2

???attribute.lit??? ???displayArticles.show???

	Fig 2. Analysis of G-V relations from Kv1.2, ÎT1-Kv1.2, K-Shaw2, K-Shaw2 T330G and ÎT1-K-Shaw2.(A) Best-fit 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 Student-t test is shown above each graph, and the red marks indicate the mean values of the sample. (B)â(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 whole-oocyte 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, anesthetic-exposed, and washout, respectively. The scale bars indicate 50 ms and 0.5 Î¼A. (B) Concentration-response relations of various general anesthetics acting on the Kv1.2 channel. Solid lines are the best fits assuming the Hill equation for sevoflurane and n-butanol (Materials and Methods). Considering the magnitude of the change and the concentrations tested, only sevoflurane and n-butanol produced reliable Hill equation fits. N = 5â7 oocytes for each concentration. Best-fit parameters are summarized in Table 1.
	Fig 4. Modulation of Kv1.2 FRAKT by general anesthetics.(A) Sequence alignment of the S4-S5 linker from K-Shaw2 (314â332) and Kv1.2 (313â331) 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 K-Shaw2 to create the Kv1.2 FRAKT mutant channel. (B) Effects of general anesthetics on whole-oocyte Kv1.2-FRAKT 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, anesthetic-exposed, and washout, respectively. The scale bars indicate 50 ms and 0.5 Î¼A. (C) Concentration-response relations of various general anesthetics acting on the Kv1.2 FRAKT channel. Solid lines are the best fits assuming the Hill equation (propofol and n-butanol) 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 = 5â8 oocytes for each concentration. Best-fit 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 whole-oocyte 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, anesthetic-exposed, and washout, respectively. The scale bars indicate 50 ms and 1 Î¼A. (B) Concentration-response 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 = 4â8 oocytes for each dose. Best-fit parameters are summarized in Table 1.
	Fig 6. Novel G-V relations of Kv1.2 FRAKT and Kv1.2 G329T in the absence and presence of sevoflurane.(A) Families of whole-oocyte 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 Î¼A. (B) Families of whole-oocyte 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 Î¼A. (C) G-V 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 best-fit parameters are summarized in Table A in S1 File.
	Fig 7. Analysis of bimodal G-V relations from Kv1.2 FRAKT, Kv1.2 G329T and ÎT1-Kv1.2 FRAKT.(A) Best-fit 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 Student-t test is shown above each graph, and the red marks indicate the mean values of the sample. (B)â(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 voltage-dependent potentiation of the K-Shaw2 conductance by sevoflurane.(A) Families of whole-oocyte K-Shaw2 (N = 4) and K-Shaw2 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 Î¼A. (B) G-V relations of K-Shaw2 (black) and K-Shaw2 T330G (red) in the absence (open) and presence of 1 mM sevoflurane (filled). Solid lines are the best-fits to the Boltzmann equation. Best-fit parameters are summarized in Fig 2 and Table A in S1 File. (C) The voltage dependence of the conductance ratio (GSevo/G0) K-Shaw2 (black) and K-Shaw2 T330G (red).
	Fig 9. Positive modulation by sevoflurane is T1 domain-independent in Kv1.2 and Kv1.2-FRAKT, and T1 domain-dependent in K-Shaw2.(A) Concentration-response relations of various general anesthetics acting on ÎT1-Kv1.2. Solid line is the best fit to the Hill equation for n-butanol. (B) Concentration-response relations of various general anesthetics acting on ÎT1-Kv1.2 FRAKT. Solid lines are the best fits to the Hill equation (propofol and n-butanol) or double Hill equation (sevoflurane, isoflurane, and halothane). Best-fit parameters for results in panels A and B are summarized in Table 1. (C) Concentration-response relations of sevoflurane acting on K-Shaw2 and ÎT1-K-Shaw2. Solid line is the best-fit double Hill equation to the K-Shaw2 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 K-Shaw2 (Table 1) [7]. K-Shaw2 and ÎT1-K-Shaw2 were tested at +60 mV. N = 2â8 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) Close-up 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 activation-open and resting-closed conformations of the channel.
	Fig 1. Positive modulation of the Kv1.2 conductance by sevoflurane.(A) Families of whole-oocyte Kv1.2 currents before (left) and after exposure to 1 mM sevoflurane (center). 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 (right) directly compares selected currents in the absence (black) and presence (red) of sevoflurane (currents evoked by steps to the indicated voltages). (B) Normalized G-V relations of Kv1.2 in the absence (black) and presence of 1 mM sevoflurane (red) (N = 6). The solid lines are the best-fit Boltzmann functions. G max is the control maximum conductance before exposure to sevoflurane (Materials and Methods). The mean best-fit parameters are summarized in Table A in S1 File. (C) The voltage dependence of the Kv1.2 conductance ratio (G Sevo/G 0). This ratio was calculated from paired measurements of the G-V relations before (G 0) and after exposure to sevolfurane (G Sevo).

References [+] :

Alkire, Thalamic microinfusion of antibody to a voltage-gated potassium channel restores consciousness during anesthesia. 2009, Pubmed