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Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel.

Vaid M, Claydon TW, Rezazadeh S, Fedida D - J. Gen. Physiol. (2008)

Bottom Line: Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4).Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening.By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

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Effect of prepulse duration on the time course of fluorescence recovery. (A and B) Ionic current and fluorescence signals recorded from TMRM-labeled Kv1.5 A397C channels during a 75-ms test pulse to +60 mV applied 7.5 ms and 37.5 ms following a +60-mV conditioning pulse of either 15 ms (A) or 5 ms (B) duration. The dotted line marks the conditioning pulse peak fluorescence amplitude for comparison.
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fig9: Effect of prepulse duration on the time course of fluorescence recovery. (A and B) Ionic current and fluorescence signals recorded from TMRM-labeled Kv1.5 A397C channels during a 75-ms test pulse to +60 mV applied 7.5 ms and 37.5 ms following a +60-mV conditioning pulse of either 15 ms (A) or 5 ms (B) duration. The dotted line marks the conditioning pulse peak fluorescence amplitude for comparison.

Mentions: The slow return of the fluorescence upon repolarization suggests that although associated with channel opening, the dequenching component of fluorescence does not reflect gating of the intracellular pore. We also observed that the recovery of this phase was dependent upon the conditioning pulse duration (Fig. 9). Following longer conditioning pulses (15 ms; Fig. 9 A) that allowed significant channel opening, the dequenching component was almost absent in the first test pulse, whereas following shorter conditioning pulses (5 ms; Fig. 9 B) that restricted channel opening, there was a robust component in the first test pulse. These data further demonstrate that the slowly recovering fluorescence dequenching is related to channel opening. Based on these data, we considered that the rapid fluorescence changes might reflect structural events occurring at the outer pore gate upon opening.


Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel.

Vaid M, Claydon TW, Rezazadeh S, Fedida D - J. Gen. Physiol. (2008)

Effect of prepulse duration on the time course of fluorescence recovery. (A and B) Ionic current and fluorescence signals recorded from TMRM-labeled Kv1.5 A397C channels during a 75-ms test pulse to +60 mV applied 7.5 ms and 37.5 ms following a +60-mV conditioning pulse of either 15 ms (A) or 5 ms (B) duration. The dotted line marks the conditioning pulse peak fluorescence amplitude for comparison.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483330&req=5

fig9: Effect of prepulse duration on the time course of fluorescence recovery. (A and B) Ionic current and fluorescence signals recorded from TMRM-labeled Kv1.5 A397C channels during a 75-ms test pulse to +60 mV applied 7.5 ms and 37.5 ms following a +60-mV conditioning pulse of either 15 ms (A) or 5 ms (B) duration. The dotted line marks the conditioning pulse peak fluorescence amplitude for comparison.
Mentions: The slow return of the fluorescence upon repolarization suggests that although associated with channel opening, the dequenching component of fluorescence does not reflect gating of the intracellular pore. We also observed that the recovery of this phase was dependent upon the conditioning pulse duration (Fig. 9). Following longer conditioning pulses (15 ms; Fig. 9 A) that allowed significant channel opening, the dequenching component was almost absent in the first test pulse, whereas following shorter conditioning pulses (5 ms; Fig. 9 B) that restricted channel opening, there was a robust component in the first test pulse. These data further demonstrate that the slowly recovering fluorescence dequenching is related to channel opening. Based on these data, we considered that the rapid fluorescence changes might reflect structural events occurring at the outer pore gate upon opening.

Bottom Line: Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4).Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening.By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

Show MeSH