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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) Representative fluorescence signals recorded from Kv1.5 A397C R487V mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or nine (W472F) other cells. (C and D) Fluorescence signals recorded from Kv1.5 A397C H463G mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or five (H463G) other cells.
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fig11: Immobilization of the selectivity filter gate abolishes the fluorescence dequenching. (A and B) Representative fluorescence signals recorded from Kv1.5 A397C R487V mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or nine (W472F) other cells. (C and D) Fluorescence signals recorded from Kv1.5 A397C H463G mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or five (H463G) other cells.

Mentions: Closure of the selectivity filter gate underlies P/C-type inactivation in Kv channels (De Biasi et al., 1993; Lopez-Barneo et al., 1993; Yellen, 1998; Kiss et al., 1999; Kurata and Fedida, 2006; Cordero-Morales et al., 2007). Although the time course of the fluorescence dequenching in Fig. 2 E is very rapid (3.4 ms at +60 mV) compared with the time course of P/C-type inactivation, which occurs in the order of seconds, we considered whether the novel fluorescence report from TMRM attached at A397C might reflect selectivity filter rearrangements that are associated in some way with inactivation. To test this, we examined the effects on the rapid component of fluorescence in Kv1.5 channels of the introduction of the outer pore mutation R487V (equivalent to T449V in Shaker), which is known to inhibit P/C-type inactivation in Shaker (Lopez-Barneo et al., 1993), and to abolish inactivation in Na+-conducting Kv1.5 channels (Wang et al., 2000a,b). Data in Fig. 11 (A and B) show a typical ionic current and fluorescence signal recorded from TMRM attached at A397C in Kv1.5 R487V mutant channels during a 100-ms voltage clamp pulse to +60 mV. The tracing clearly shows that the dequenching component of fluorescence is maintained in the R487V mutation.


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) Representative fluorescence signals recorded from Kv1.5 A397C R487V mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or nine (W472F) other cells. (C and D) Fluorescence signals recorded from Kv1.5 A397C H463G mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or five (H463G) other cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig11: Immobilization of the selectivity filter gate abolishes the fluorescence dequenching. (A and B) Representative fluorescence signals recorded from Kv1.5 A397C R487V mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or nine (W472F) other cells. (C and D) Fluorescence signals recorded from Kv1.5 A397C H463G mutant channels during a 100-ms voltage clamp pulse from −80 to +60 mV. Similar recordings were obtained from four (R487V) or five (H463G) other cells.
Mentions: Closure of the selectivity filter gate underlies P/C-type inactivation in Kv channels (De Biasi et al., 1993; Lopez-Barneo et al., 1993; Yellen, 1998; Kiss et al., 1999; Kurata and Fedida, 2006; Cordero-Morales et al., 2007). Although the time course of the fluorescence dequenching in Fig. 2 E is very rapid (3.4 ms at +60 mV) compared with the time course of P/C-type inactivation, which occurs in the order of seconds, we considered whether the novel fluorescence report from TMRM attached at A397C might reflect selectivity filter rearrangements that are associated in some way with inactivation. To test this, we examined the effects on the rapid component of fluorescence in Kv1.5 channels of the introduction of the outer pore mutation R487V (equivalent to T449V in Shaker), which is known to inhibit P/C-type inactivation in Shaker (Lopez-Barneo et al., 1993), and to abolish inactivation in Na+-conducting Kv1.5 channels (Wang et al., 2000a,b). Data in Fig. 11 (A and B) show a typical ionic current and fluorescence signal recorded from TMRM attached at A397C in Kv1.5 R487V mutant channels during a 100-ms voltage clamp pulse to +60 mV. The tracing clearly shows that the dequenching component of fluorescence is maintained in the R487V mutation.

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