Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 1. Comparison of WT hERG gating and ionic currents over 24 ms and 300 ms depolarizing pulses. (A) Representative traces of gating currents recorded in response to depolarizing steps of 24 ms from a HP of -110 mV. Traces from depolarizations between -60 and +60 are shown in 20 mV increments. Inset are representative traces of ionic currents recorded using the same protocol illustrating traces from depolarizations between -90 and +190 in 20 mV increments. (B) Isolated traces of gating currents demonstrate a fast IgON component of charge movement at all voltages and the emergence of a slower component of charge movement positive to 20 mV which is clear in the biexponential fit of the IgOFF currents at +60 mV. (C) Representative gating (from -60 to +60 mV) and ionic (inset, from -60 to +80 mV) current traces from a family of 300 ms depolarizing pulses. (D) Isolated gating current traces illustrating the development of a slow IgON component which develops concurrently with a slowing in the IgOFF currents. (E) Isochronal peak ionic tail current GV relationships for 24 ms (○) and 300 ms (∆) depolarizations and QOFF-V relationships from integration of IgOFF currents for 24 ms (●) and 300 ms (▲) depolarizations. Data points were fit with a single Boltzmann function of the form  where y/ymax is the normalized response; either G/Gmax or Q/Qmax, V0.5 the half activation potential and k the slope factor or a double Boltzmann function  where A is the amplitude of the fit component.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048346&req=5

Figure 1: Figure 1. Comparison of WT hERG gating and ionic currents over 24 ms and 300 ms depolarizing pulses. (A) Representative traces of gating currents recorded in response to depolarizing steps of 24 ms from a HP of -110 mV. Traces from depolarizations between -60 and +60 are shown in 20 mV increments. Inset are representative traces of ionic currents recorded using the same protocol illustrating traces from depolarizations between -90 and +190 in 20 mV increments. (B) Isolated traces of gating currents demonstrate a fast IgON component of charge movement at all voltages and the emergence of a slower component of charge movement positive to 20 mV which is clear in the biexponential fit of the IgOFF currents at +60 mV. (C) Representative gating (from -60 to +60 mV) and ionic (inset, from -60 to +80 mV) current traces from a family of 300 ms depolarizing pulses. (D) Isolated gating current traces illustrating the development of a slow IgON component which develops concurrently with a slowing in the IgOFF currents. (E) Isochronal peak ionic tail current GV relationships for 24 ms (○) and 300 ms (∆) depolarizations and QOFF-V relationships from integration of IgOFF currents for 24 ms (●) and 300 ms (▲) depolarizations. Data points were fit with a single Boltzmann function of the form where y/ymax is the normalized response; either G/Gmax or Q/Qmax, V0.5 the half activation potential and k the slope factor or a double Boltzmann function where A is the amplitude of the fit component.

Mentions: Using the COVG clamp we recorded hERG gating currents using a 24 ms protocol as shown in Figure 1A and B. We observed a fast component that moved at negative potentials with a fast return upon repolarization to -110 mV as illustrated by the fast decaying ON gating currents (IgON) and OFF gating currents (IgOFF) displayed in the isolated -20 mV trace. During stronger depolarizations a slower component in IgOFF emerged and the current was best fit by a two component exponential with time constants of ~0.5 and 8 ms. It is difficult to accurately resolve charge by integrating slow gating transitions that produce small IgON. Therefore, it is common to assess the amount of charge moved in a given conditioning pulse by integrating the IgOFF upon repolarization as OFF gating currents are typically faster as voltage sensors return from activated positions. The Qoff measurement was therefore used to plot the Qoff-V shown in Figure 1E (●). The data was best fit with a double component Boltzmann equation with parameters V0.5a = -14.2 ± 0.2 mV, ka = 9.4 ± 1.7 mV, Aa = 0.31 ± 0.04, V0.5b = 41.2 ± 1.9 mV, kb = 13.1 ± 1.1 mV (n = 3), where Aa is the amplitude of the first component fit which represents the faster charge system that appears before the emergence of the slower charge system, and at more negative potentials.


Gating charge movement precedes ionic current activation in hERG channels.

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

Figure 1. Comparison of WT hERG gating and ionic currents over 24 ms and 300 ms depolarizing pulses. (A) Representative traces of gating currents recorded in response to depolarizing steps of 24 ms from a HP of -110 mV. Traces from depolarizations between -60 and +60 are shown in 20 mV increments. Inset are representative traces of ionic currents recorded using the same protocol illustrating traces from depolarizations between -90 and +190 in 20 mV increments. (B) Isolated traces of gating currents demonstrate a fast IgON component of charge movement at all voltages and the emergence of a slower component of charge movement positive to 20 mV which is clear in the biexponential fit of the IgOFF currents at +60 mV. (C) Representative gating (from -60 to +60 mV) and ionic (inset, from -60 to +80 mV) current traces from a family of 300 ms depolarizing pulses. (D) Isolated gating current traces illustrating the development of a slow IgON component which develops concurrently with a slowing in the IgOFF currents. (E) Isochronal peak ionic tail current GV relationships for 24 ms (○) and 300 ms (∆) depolarizations and QOFF-V relationships from integration of IgOFF currents for 24 ms (●) and 300 ms (▲) depolarizations. Data points were fit with a single Boltzmann function of the form  where y/ymax is the normalized response; either G/Gmax or Q/Qmax, V0.5 the half activation potential and k the slope factor or a double Boltzmann function  where A is the amplitude of the fit component.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048346&req=5

Figure 1: Figure 1. Comparison of WT hERG gating and ionic currents over 24 ms and 300 ms depolarizing pulses. (A) Representative traces of gating currents recorded in response to depolarizing steps of 24 ms from a HP of -110 mV. Traces from depolarizations between -60 and +60 are shown in 20 mV increments. Inset are representative traces of ionic currents recorded using the same protocol illustrating traces from depolarizations between -90 and +190 in 20 mV increments. (B) Isolated traces of gating currents demonstrate a fast IgON component of charge movement at all voltages and the emergence of a slower component of charge movement positive to 20 mV which is clear in the biexponential fit of the IgOFF currents at +60 mV. (C) Representative gating (from -60 to +60 mV) and ionic (inset, from -60 to +80 mV) current traces from a family of 300 ms depolarizing pulses. (D) Isolated gating current traces illustrating the development of a slow IgON component which develops concurrently with a slowing in the IgOFF currents. (E) Isochronal peak ionic tail current GV relationships for 24 ms (○) and 300 ms (∆) depolarizations and QOFF-V relationships from integration of IgOFF currents for 24 ms (●) and 300 ms (▲) depolarizations. Data points were fit with a single Boltzmann function of the form where y/ymax is the normalized response; either G/Gmax or Q/Qmax, V0.5 the half activation potential and k the slope factor or a double Boltzmann function where A is the amplitude of the fit component.
Mentions: Using the COVG clamp we recorded hERG gating currents using a 24 ms protocol as shown in Figure 1A and B. We observed a fast component that moved at negative potentials with a fast return upon repolarization to -110 mV as illustrated by the fast decaying ON gating currents (IgON) and OFF gating currents (IgOFF) displayed in the isolated -20 mV trace. During stronger depolarizations a slower component in IgOFF emerged and the current was best fit by a two component exponential with time constants of ~0.5 and 8 ms. It is difficult to accurately resolve charge by integrating slow gating transitions that produce small IgON. Therefore, it is common to assess the amount of charge moved in a given conditioning pulse by integrating the IgOFF upon repolarization as OFF gating currents are typically faster as voltage sensors return from activated positions. The Qoff measurement was therefore used to plot the Qoff-V shown in Figure 1E (●). The data was best fit with a double component Boltzmann equation with parameters V0.5a = -14.2 ± 0.2 mV, ka = 9.4 ± 1.7 mV, Aa = 0.31 ± 0.04, V0.5b = 41.2 ± 1.9 mV, kb = 13.1 ± 1.1 mV (n = 3), where Aa is the amplitude of the first component fit which represents the faster charge system that appears before the emergence of the slower charge system, and at more negative potentials.

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
Related in: MedlinePlus