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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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Immobilization of the selectivity filter gate abolishes the fluorescence dequenching. (A and B) Ionic current and fluorescence signals recorded from Kv1.5 A397C channels during 100-ms pulses to +60 mV in the presence of 3 and 99 mM external K+. (C) Mean F-V relation with 99 mM K+ plotted alongside the G-V and F-V relations obtained with 3 mM K+ to demonstrate the voltage dependence of voltage sensor movement when the selectivity filter gate is immobilized by high external K+. The G-V relation with 99 mM K+ is not shown because of the large error associated with calculations around the reversal potential (∼0 mV). V1/2 and k values for the F-V relation with 99 mM K+ were −36.4 ± 1.2 and 12.8 ± 1.0 mV, respectively (n = 3). V1/2 and k values for the G-V and F-V relations with 3 mM K+ were 8.1 ± 2.5 and 18.3 ± 1.7 mV (G-V) and 4.3 ± 2.0 and 21.5 ± 1.3 mV (F-V), respectively (n = 3). The F-V relation with high external K+ was therefore 40 mV left shifted from the G-V relation. (D) Ionic current and fluorescence signals from Kv1.5 A397C W472F mutant channels (W472F is equivalent to the W434F mutation in Shaker channels) during a 100-ms voltage clamp pulse from −80 to +60 mV. Note that only small leak currents were observed from oocytes injected with Kv1.5 A397C W472F. Similar recordings were obtained from nine (W472F) other cells.
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fig10: Immobilization of the selectivity filter gate abolishes the fluorescence dequenching. (A and B) Ionic current and fluorescence signals recorded from Kv1.5 A397C channels during 100-ms pulses to +60 mV in the presence of 3 and 99 mM external K+. (C) Mean F-V relation with 99 mM K+ plotted alongside the G-V and F-V relations obtained with 3 mM K+ to demonstrate the voltage dependence of voltage sensor movement when the selectivity filter gate is immobilized by high external K+. The G-V relation with 99 mM K+ is not shown because of the large error associated with calculations around the reversal potential (∼0 mV). V1/2 and k values for the F-V relation with 99 mM K+ were −36.4 ± 1.2 and 12.8 ± 1.0 mV, respectively (n = 3). V1/2 and k values for the G-V and F-V relations with 3 mM K+ were 8.1 ± 2.5 and 18.3 ± 1.7 mV (G-V) and 4.3 ± 2.0 and 21.5 ± 1.3 mV (F-V), respectively (n = 3). The F-V relation with high external K+ was therefore 40 mV left shifted from the G-V relation. (D) Ionic current and fluorescence signals from Kv1.5 A397C W472F mutant channels (W472F is equivalent to the W434F mutation in Shaker channels) during a 100-ms voltage clamp pulse from −80 to +60 mV. Note that only small leak currents were observed from oocytes injected with Kv1.5 A397C W472F. Similar recordings were obtained from nine (W472F) other cells.

Mentions: There is strong evidence for a K+ modulatory site within the outer pore of Kv channels that, when occupied, prevents collapse of the selectivity filter, acting with a “foot-in-the-door” mechanism (Pardo et al., 1992; Lopez-Barneo et al., 1993; Baukrowitz and Yellen, 1995; Rasmusson et al., 1995; Kiss and Korn, 1998). In some Kv1 channels, including Kv1.5 (Fedida et al., 1999), this regulation does not appear to be as strong as in Shaker channels, but raising the extracellular K+ did significantly modulate the fluorescence report (Fig. 10). The ionic current amplitudes and time course are relatively unchanged at +60 mV, despite the reduction in driving force, as reported before for some Kv1 channels (Pardo et al., 1992). The fluorescence signals in Fig. 10 B show that raising the external K+ concentration to 99 mM abolished the dequenching component and restored more of a Shaker-like fluorescence report of voltage sensor movement. The fluorescence report is not identical to that in Shaker (Fig. 2), though, and appears to show both a fast and slow component of quenching, but with 99 mM K+ the left-shifted F-V relation of voltage sensor movement (V1/2 = −36.4 ± 1.2 mV) becomes apparent (Fig. 10 C; n = 3). Clearly, K+ regulation at an outer pore site is able to modulate the fluorescence environment change detected at A359C, and allow detection of voltage sensor movement.


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)

Immobilization of the selectivity filter gate abolishes the fluorescence dequenching. (A and B) Ionic current and fluorescence signals recorded from Kv1.5 A397C channels during 100-ms pulses to +60 mV in the presence of 3 and 99 mM external K+. (C) Mean F-V relation with 99 mM K+ plotted alongside the G-V and F-V relations obtained with 3 mM K+ to demonstrate the voltage dependence of voltage sensor movement when the selectivity filter gate is immobilized by high external K+. The G-V relation with 99 mM K+ is not shown because of the large error associated with calculations around the reversal potential (∼0 mV). V1/2 and k values for the F-V relation with 99 mM K+ were −36.4 ± 1.2 and 12.8 ± 1.0 mV, respectively (n = 3). V1/2 and k values for the G-V and F-V relations with 3 mM K+ were 8.1 ± 2.5 and 18.3 ± 1.7 mV (G-V) and 4.3 ± 2.0 and 21.5 ± 1.3 mV (F-V), respectively (n = 3). The F-V relation with high external K+ was therefore 40 mV left shifted from the G-V relation. (D) Ionic current and fluorescence signals from Kv1.5 A397C W472F mutant channels (W472F is equivalent to the W434F mutation in Shaker channels) during a 100-ms voltage clamp pulse from −80 to +60 mV. Note that only small leak currents were observed from oocytes injected with Kv1.5 A397C W472F. Similar recordings were obtained from nine (W472F) other cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2483330&req=5

fig10: Immobilization of the selectivity filter gate abolishes the fluorescence dequenching. (A and B) Ionic current and fluorescence signals recorded from Kv1.5 A397C channels during 100-ms pulses to +60 mV in the presence of 3 and 99 mM external K+. (C) Mean F-V relation with 99 mM K+ plotted alongside the G-V and F-V relations obtained with 3 mM K+ to demonstrate the voltage dependence of voltage sensor movement when the selectivity filter gate is immobilized by high external K+. The G-V relation with 99 mM K+ is not shown because of the large error associated with calculations around the reversal potential (∼0 mV). V1/2 and k values for the F-V relation with 99 mM K+ were −36.4 ± 1.2 and 12.8 ± 1.0 mV, respectively (n = 3). V1/2 and k values for the G-V and F-V relations with 3 mM K+ were 8.1 ± 2.5 and 18.3 ± 1.7 mV (G-V) and 4.3 ± 2.0 and 21.5 ± 1.3 mV (F-V), respectively (n = 3). The F-V relation with high external K+ was therefore 40 mV left shifted from the G-V relation. (D) Ionic current and fluorescence signals from Kv1.5 A397C W472F mutant channels (W472F is equivalent to the W434F mutation in Shaker channels) during a 100-ms voltage clamp pulse from −80 to +60 mV. Note that only small leak currents were observed from oocytes injected with Kv1.5 A397C W472F. Similar recordings were obtained from nine (W472F) other cells.
Mentions: There is strong evidence for a K+ modulatory site within the outer pore of Kv channels that, when occupied, prevents collapse of the selectivity filter, acting with a “foot-in-the-door” mechanism (Pardo et al., 1992; Lopez-Barneo et al., 1993; Baukrowitz and Yellen, 1995; Rasmusson et al., 1995; Kiss and Korn, 1998). In some Kv1 channels, including Kv1.5 (Fedida et al., 1999), this regulation does not appear to be as strong as in Shaker channels, but raising the extracellular K+ did significantly modulate the fluorescence report (Fig. 10). The ionic current amplitudes and time course are relatively unchanged at +60 mV, despite the reduction in driving force, as reported before for some Kv1 channels (Pardo et al., 1992). The fluorescence signals in Fig. 10 B show that raising the external K+ concentration to 99 mM abolished the dequenching component and restored more of a Shaker-like fluorescence report of voltage sensor movement. The fluorescence report is not identical to that in Shaker (Fig. 2), though, and appears to show both a fast and slow component of quenching, but with 99 mM K+ the left-shifted F-V relation of voltage sensor movement (V1/2 = −36.4 ± 1.2 mV) becomes apparent (Fig. 10 C; n = 3). Clearly, K+ regulation at an outer pore site is able to modulate the fluorescence environment change detected at A359C, and allow detection of voltage sensor movement.

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
Related in: MedlinePlus