Open-channel blocking action of volatile anaesthetics desflurane and sevoflurane on human voltage-gated Kv 1.5 channel

Abstract

Background and purpose: Volatile anaesthetics have been shown to differentially modulate mammalian Shaker-related voltage-gated potassium (K_v 1.x) channels. This study was designed to investigate molecular and cellular mechanisms underlying the modulatory effects of desflurane or sevoflurane on human K_v 1.5 (hK_v 1.5) channels.

Experimental approach: Thirteen single-point mutations were constructed within pore domain of hK_v 1.5 channel using site-directed mutagenesis. The effects of desflurane or sevoflurane on heterologously expressed wild-type and mutant hK_v 1.5 channels were examined by whole-cell patch-clamp technique. A computer simulation was conducted to predict the docking pose of desflurane or sevoflurane within hK_v 1.5 channel.

Key results: Both desflurane and sevoflurane increased hK_v 1.5 current at mild depolarizations but decreased it at strong depolarizations, indicating that these anaesthetics produce both stimulatory and inhibitory actions on hK_v 1.5 channels. The inhibitory effect of desflurane or sevoflurane on hK_v 1.5 channels arose primarily from its open-channel blocking action. The inhibitory action of desflurane or sevoflurane on hK_v 1.5 channels was significantly attenuated in T480A, V505A, and I508A mutant channels, compared with wild-type channel. Computational docking simulation predicted that desflurane or sevoflurane resides within the inner cavity of channel pore and has contact with Thr479, Thr480, Val505, and Ile508.

Conclusion and implications: Desflurane and sevoflurane exert an open-channel blocking action on hK_v 1.5 channels by functionally interacting with specific amino acids located within the channel pore. This study thus identifies a novel molecular basis mediating inhibitory modulation of hK_v 1.5 channels by desflurane and sevoflurane.

FIGURE 1

Effect of desflurane on hK_v1.5 channels heterologously expressed in CHO cells. (a) Superimposed traces of hK_v1.5 currents activated during 300‐ms depolarizing voltage‐clamp steps to test potentials of −50 to +50 mV in 10‐mV steps applied from a holding potential of −80 mV, before (left panel) and 7 min after exposure to 18% desflurane (right panel). Voltage‐clamp protocol is illustrated above the control traces of hK_v1.5 current. (b) Current–voltage relationships for the initial (left panel) and late current levels (right panel) measured during depolarizing steps in the absence (Control) and presence of 18% desflurane. Amplitude of initial or late current at each test potential was normalized with reference to its initial current amplitude measured at +50 mV in control (n = 6). (c) Current ratio of initial and late currents, obtained by dividing the current amplitude in the presence of desflurane by that in its absence at each test potential, in the experimental results shown in panel (b). The dashed line indicates I _Desflurane/I _Control = 1. Note that while the initial current ratio was more than 1 at potentials between −40 and 0 mV, the late current ratio was more than 1 at potentials between −40 and −20 mV. *P < 0.05 between the initial and late current ratios. (d) The current–voltage relationships for the tail currents of hK_v1.5 channels measured upon repolarization to −40 mV from various test potentials in the absence and presence of 18% desflurane. The smooth curves through the data points represent least‐squares fittings of the Boltzmann equation to the data points, yielding V _h (V _h, −12.5 ± 5.0 mV in control; −30.0 ± 1.2 mV in the presence of 18% desflurane, n = 6)

FIGURE 2

Open‐channel blocking effect of desflurane on hK_v1.5 channels. (a) Superimposed hK_v1.5 currents recorded during 300‐ms depolarizing steps to +30 mV before and during exposure to 18% desflurane. (b) Time constant (τ) for the hK_v1.5 current decline during 300‐ms depolarizing steps in the absence (Control) and presence of 18% desflurane, obtained by least‐squares fitting of the single exponential function. *P < 0.05 between control and 18% desflurane groups (n = 6). (c) The current ratio (I _Desflurane/I _Control) obtained by dividing hK_v1.5 current in the presence of 18% desflurane (I _Desflurane) by that in its absence (I _Control), shown in Figure 2a. The smooth curve through the data points (red) represents a least‐squares fit of a single exponential function, yielding a time constant for channel block (τ). (d) Time constant (τ) for hK_v1.5 channel inhibition by desflurane at various test potentials (n = 6). *P < 0.05, compared with τ at −10 mV. (e) Superimposed tail currents elicited upon repolarization to −40 mV following depolarizing steps to +30 mV in the absence (Control) and presence of 18% desflurane. The arrow shows crossover of tail currents. (f) Time constant (τ) for the decay of tail currents in the absence (Control) and presence of 18% desflurane, obtained by a least‐squares fit of the single exponential function. *P < 0.05 between control and 18% desflurane groups (n = 6)

FIGURE 3

Reversibility and concentration dependence of desflurane‐induced inhibition of hK_v1.5 current. (a) Changes in the late current amplitude of hK_v1.5 current, activated by 300‐ms depolarizing step to +30 mV every 10 s, before, during exposure to 18% desflurane, and after its washout. The desflurane‐induced reduction of hK_v1.5 current reached a maximum approximately 7 min after the application of desflurane, and then it was completely recovered to the control level after the washout of 18% desflurane. The horizontal bar indicates the period of desflurane application. The inset (right panel) shows the original current traces obtained at the time points indicated by numerals. (b) Superimposed traces of hK_v1.5 current recorded during 300‐ms depolarizing step to +30 mV before and during exposure to increasing concentrations of desflurane (6%, 12%, and 18%) applied in a cumulative manner. A higher concentration of desflurane was applied after the response to the previous concentration reached a steady state (usually 3 min). (c) Mean concentration–response relationships for the inhibition of hK_v1.5 current by desflurane. The data points (n = 6) represent the percent inhibition of late current, calculated as the reduction with respect to control measured at the end of 300‐ms depolarizing step to +30 mV and fitted with the Hill equation. The concentration of desflurane is shown in the millimolar order

FIGURE 4

Effect of 18% desflurane on wild‐type (WT) and mutant hK_v1.5 channel currents. (a) Superimposed current traces recorded from WT and 13 mutant hK_v1.5 channels (T462C, H463C, T479A, T480A, R487V, A501V, I502A, V505A, I508A, A509G, L510A, V512A, and V516A) during 300‐ms depolarizing steps to +30 mV in the absence (open square) and presence (filled square) of 18% desflurane. (b) Percent change in the late current amplitude at +30 mV caused by 18% desflurane in WT and 13 mutant hK_v1.5 channel currents. *P < 0.05, compared with WT. n = 6 in each group

FIGURE 5

The concentration dependence of desflurane‐induced inhibition in mutant hK_v1.5 channels. (a) Superimposed current traces recorded from mutant (T480A, V505A, I508A, and A509G) hK_v1.5 channels during 300‐ms depolarizing step to +30 mV in the absence and presence of 6%, 12%, and 18% desflurane applied in a cumulative way. (b) The mean concentration–response relationships for the inhibition of late current at +30 mV in wild‐type (WT) and four mutant (T480A, V505A, I508A, and A509G) hK_v1.5 channels. The dashed curve (WT) and continuous curves (T480A, V505A, I508A, and A509G) through data points represent a least‐squares fit of Hill equation, yielding IC₅₀ values. (c) IC₅₀ values for WT and mutant (T480A, V505A, I508A, and A509G) hKv1.5 channels. The desflurane concentration is expressed as millimolar order in panels (b) and (c). *P < 0.05, compared with WT. n = 6 in each group

FIGURE 6

Modulatory effects of sevoflurane on hK_v1.5 current. (a) Superimposed traces of hK_v1.5 currents activated during 300‐ms depolarizing steps to test potentials of −50 to +50 mV in 10 mV steps applied from a holding potential of −80 mV, before (left panel, Control) and 6 min after exposure to 8% sevoflurane (right panel). The voltage‐clamp protocol is illustrated above the control traces of hKv1.5 current. (b) The current–voltage relationships for the initial (left panel) and late current levels (right panel) measured during depolarizing steps in control and in the presence of 8% sevoflurane. The amplitude of initial or late current at each test potential was normalized with reference to its initial current amplitude measured at +50 mV in control (n = 6). (c) The ratio of initial or late current amplitudes measured in the absence and presence of 8% sevoflurane, obtained by dividing the current amplitude in the presence of sevoflurane by that in its absence at each test potential (I _Sevoflurane/I _Control). The dashed line indicates I _Sevoflurane/I _Control = 1. *P < 0.05 between the ratio of initial and late current amplitudes at each test potential. (d) The current–voltage relationships for the tail currents of hKv1.5 channel in the absence and presence of 8% sevoflurane. The smooth curves through the data points represent least‐squares fittings of the Boltzmann equation to the data points, yielding V _h. (e) V _h for the voltage‐dependent activation of hK_v1.5 current in control and in the presence of 8% sevoflurane. *P < 0.05, compared with control. n = 6 in each group. (f) Mean concentration–response relationships for the inhibition of hK_v1.5 current by sevoflurane. The data points (n = 6) represent the percent inhibition of late current, calculated as the reduction with respect to control value measured at the end of 300‐ms depolarizing step to +30 mV. The smooth curve through the data points represents a least‐squares fit of Hill equation, yielding an IC₅₀ of 1.01 ± 0.14 mM, which corresponds to 6.9 ± 1.0% (n = 6). The concentration of sevoflurane is shown as millimolar order. Inset, superimposed traces of hK_v1.5 current recorded during 300‐ms depolarizing step to +30 mV before and during exposure to increasing concentrations (2%, 4%, 6%, and 8%) of sevoflurane applied in a cumulative manner

FIGURE 7

Open‐channel blocking effect of sevoflurane on hK_v1.5 channels. (a) Superimposed hK_v1.5 currents recorded during 300‐ms depolarizing step to +30 mV before and during exposure to 8% sevoflurane. (b) Time constant (τ) for the hK_v1.5 current decline during 300‐ms depolarizing steps in the absence (Control) and presence of 8% sevoflurane, obtained by least‐squares fitting of single exponential function. *P < 0.05 between control and 8% sevoflurane groups (n = 6). (c) The current ratio (I _Sevoflurane/I _Control) obtained by dividing hK_v1.5 current in the presence of 8% sevoflurane (I _Sevoflurane) by that in its absence (I _Control), shown in Figure 7a. The smooth curve through the data points (red) represents a least‐squares fit of a single exponential function, yielding a time constant for channel block (τ). (d) Time constant (τ) for hK_v1.5 channel inhibition by sevoflurane at various test potentials (n = 6). *P < 0.05, compared with τ at −10 mV. (e) Superimposed tail currents elicited upon repolarization to −40 mV following depolarizing steps to +30 mV in the absence (Control) and presence of 8% sevoflurane. The arrow shows crossover of tail currents. (f) The time constant (τ) for the decay of tail currents in the absence and presence of 8% sevoflurane, obtained by the least‐squares fit of the single exponential function. *P < 0.05 between control and 8% sevoflurane groups (n = 6)

FIGURE 8

Effect of 8% sevoflurane on wild‐type (WT) and mutant hK_v1.5 channel currents. (a) Superimposed current traces recorded from WT and 13 mutant hK_v1.5 channels (T462C, H463C, T479A, T480A, R487V, A501V, I502A, V505A, I508A, A509G, L510A, V512A, and V516A) during 300‐ms depolarizing step to +30 mV in the absence (open square) and presence (filled square) of 8% sevoflurane. (b) The percent change in the late current amplitude at +30 mV caused by 8% sevoflurane in WT and 13 mutant hKv1.5 channel currents. *P < 0.05, compared with WT. n = 6 in each group

FIGURE 9

A simulated docking pose of desflurane or sevoflurane within the pore region of the open‐state model of hK_v1.5 channel. (a and b) A side view (a) and expanded version (b) of the docked desflurane within the inner cavity of the hK_v1.5 channel pore. (c and d) A side view (c) and expanded version (d) of the docked sevoflurane within the inner cavity of hK_v1.5 channel pore. Desflurane or sevoflurane was located near the base of the ion selectivity filter with the lowest docking score in the docking simulation limited within the channel pore region. Only three pore domains are shown for clarity. Desflurane and sevoflurane are shown as a space‐filled model, and the amino acids predicted to reside within 4.5 Å from anaesthetics (Thr479, Thr480, Val505, and Ile508) are shown in a stick format

FIGURE 10

Changes in binding free energies by in silico alanine scanning mutagenesis in hK_v1.5 channel. (a and b) Pore domains of hK_v1.5 channel with docked desflurane (a) or sevoflurane (b) at the lowest docking score in the docking simulation limited within the channel pore region, as viewed from the intracellular side. Desflurane and sevoflurane are shown as a space‐filled model, and the amino acids predicted to reside within 4.5 Å from ligand (Thr479, Thr480, Val505, and Ile508) are shown in a stick format. (c and d) Differences in the binding free energy (ΔΔG) for desflurane (c) or sevoflurane (d), calculated before and after in silico alanine scanning mutagenesis of Thr479, Thr480, Val505, and Ile508 in hK_v1.5 channel