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Gating charge movement precedes ionic current activation in hERG channels.

Goodchild SJ, Fedida D - Channels (Austin) (2013)

Bottom Line: We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component.Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV.The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology and Therapeutics; University of British Columbia; Vancouver, BC Canada.

ABSTRACT
We recently reported gating currents recorded from hERG channels expressed in mammalian TSA cells and assessed the kinetics at different voltages. We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component. Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV. The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.

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Figure 2. Kinetics of hERG charge movement and ionic activation at 0 mV and +60 mV. Representative trace of gating currents evoked by steps to (A) 0 mV extending in 50 ms increments or (B) +60 mV in 10 ms increments. (C) Representative trace of ionic currents evoked by steps to 0 mV extending in 60 ms increments or (D) +60 mV in 10 ms increments. Plot of the normalized Qoff (●) and normalized peak ionic tail currents (○) after increasing durations of a 0 mV (E) or +60 mV (F) conditioning step. Charge movement was fit with a single exponential () and ionic currents with a delayed exponential  where y is the normalized response, t is the duration of the conditioning pulse, A the amplitude, τ the time constant and H the unit step function where t0 is the delay before onset of the exponential rise. (G) Time constants for gating charge movement and ionic current activation at 0 and +60 mV.
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Figure 2: Figure 2. Kinetics of hERG charge movement and ionic activation at 0 mV and +60 mV. Representative trace of gating currents evoked by steps to (A) 0 mV extending in 50 ms increments or (B) +60 mV in 10 ms increments. (C) Representative trace of ionic currents evoked by steps to 0 mV extending in 60 ms increments or (D) +60 mV in 10 ms increments. Plot of the normalized Qoff (●) and normalized peak ionic tail currents (○) after increasing durations of a 0 mV (E) or +60 mV (F) conditioning step. Charge movement was fit with a single exponential () and ionic currents with a delayed exponential where y is the normalized response, t is the duration of the conditioning pulse, A the amplitude, τ the time constant and H the unit step function where t0 is the delay before onset of the exponential rise. (G) Time constants for gating charge movement and ionic current activation at 0 and +60 mV.

Mentions: Data in Figure 2 shows overlain traces of gating currents in response to conditioning pulses of increasing duration at 0 mV (Fig. 2A) and +60 mV (Fig. 2B) and ionic currents at 0 mV (Fig. 2C) and +60 mV (Fig. 2D). Figure 2E and F show the normalized Qoff and peak ionic tail currents plotted against pulse duration at 0 mV and +60 mV. The development of Qoff was fit with a single exponential function at 0 mV (Fig. 2E, ●) (τ = 88.8 ± 13.7 ms [n = 5]) and at +60 mV (Fig. 2F, ●) (τ = 30.1 ± 3 ms [n = 5]). It should be noted that the time course of the development of the fast IgON is not captured in these protocols as the minimum duration pulse applied was greater than the duration of the fast IgON. Ionic currents exhibited further latency to opening which results from the transitioning of multiple closed states before opening,14 even after movement of the VSD’s to their activated position. To account for this latency the data were fit with an exponential function with a delay (τ0) at 0 mV (Fig. 2E, ○) (τ = 287 ± 23 ms, τ0 = 12 ± 4 ms [n = 4]) and +60 mV (Fig. 2F, ○) (τ = 42 ± 4 ms, τ0 = 12 ± 0.5 ms [n = 4]). The voltage dependence of the time constants is compared in Figure 2G which, in combination with the plotted data, shows clearly that gating current saturates and thus moves more quickly than channels activate at both 0 mV and +60 mV, suggesting that the bulk of gating charge moves before the channel pore opens.


Gating charge movement precedes ionic current activation in hERG channels.

Goodchild SJ, Fedida D - Channels (Austin) (2013)

Figure 2. Kinetics of hERG charge movement and ionic activation at 0 mV and +60 mV. Representative trace of gating currents evoked by steps to (A) 0 mV extending in 50 ms increments or (B) +60 mV in 10 ms increments. (C) Representative trace of ionic currents evoked by steps to 0 mV extending in 60 ms increments or (D) +60 mV in 10 ms increments. Plot of the normalized Qoff (●) and normalized peak ionic tail currents (○) after increasing durations of a 0 mV (E) or +60 mV (F) conditioning step. Charge movement was fit with a single exponential () and ionic currents with a delayed exponential  where y is the normalized response, t is the duration of the conditioning pulse, A the amplitude, τ the time constant and H the unit step function where t0 is the delay before onset of the exponential rise. (G) Time constants for gating charge movement and ionic current activation at 0 and +60 mV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Figure 2. Kinetics of hERG charge movement and ionic activation at 0 mV and +60 mV. Representative trace of gating currents evoked by steps to (A) 0 mV extending in 50 ms increments or (B) +60 mV in 10 ms increments. (C) Representative trace of ionic currents evoked by steps to 0 mV extending in 60 ms increments or (D) +60 mV in 10 ms increments. Plot of the normalized Qoff (●) and normalized peak ionic tail currents (○) after increasing durations of a 0 mV (E) or +60 mV (F) conditioning step. Charge movement was fit with a single exponential () and ionic currents with a delayed exponential where y is the normalized response, t is the duration of the conditioning pulse, A the amplitude, τ the time constant and H the unit step function where t0 is the delay before onset of the exponential rise. (G) Time constants for gating charge movement and ionic current activation at 0 and +60 mV.
Mentions: Data in Figure 2 shows overlain traces of gating currents in response to conditioning pulses of increasing duration at 0 mV (Fig. 2A) and +60 mV (Fig. 2B) and ionic currents at 0 mV (Fig. 2C) and +60 mV (Fig. 2D). Figure 2E and F show the normalized Qoff and peak ionic tail currents plotted against pulse duration at 0 mV and +60 mV. The development of Qoff was fit with a single exponential function at 0 mV (Fig. 2E, ●) (τ = 88.8 ± 13.7 ms [n = 5]) and at +60 mV (Fig. 2F, ●) (τ = 30.1 ± 3 ms [n = 5]). It should be noted that the time course of the development of the fast IgON is not captured in these protocols as the minimum duration pulse applied was greater than the duration of the fast IgON. Ionic currents exhibited further latency to opening which results from the transitioning of multiple closed states before opening,14 even after movement of the VSD’s to their activated position. To account for this latency the data were fit with an exponential function with a delay (τ0) at 0 mV (Fig. 2E, ○) (τ = 287 ± 23 ms, τ0 = 12 ± 4 ms [n = 4]) and +60 mV (Fig. 2F, ○) (τ = 42 ± 4 ms, τ0 = 12 ± 0.5 ms [n = 4]). The voltage dependence of the time constants is compared in Figure 2G which, in combination with the plotted data, shows clearly that gating current saturates and thus moves more quickly than channels activate at both 0 mV and +60 mV, suggesting that the bulk of gating charge moves before the channel pore opens.

Bottom Line: We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component.Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV.The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology and Therapeutics; University of British Columbia; Vancouver, BC Canada.

ABSTRACT
We recently reported gating currents recorded from hERG channels expressed in mammalian TSA cells and assessed the kinetics at different voltages. We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component. Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV. The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.

Show MeSH