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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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Prevention of opening abolishes the dequenching of fluorescence. (A and B) Ionic (A) and fluorescence (B) signals recorded from TMRM attached at A397C in control conditions and in the presence of 10 mM 4-AP during 100-ms voltage clamp pulses from −80 to +60 mV. 10 mM 4-AP prevented channel opening and reduced current amplitude by 66 ± 6% (n = 3). (C) Mean G-V and F-V relations in the presence of 10 mM 4-AP. V1/2 and k values for G-V and F-V relations were 44.0 ± 2.8 and 23.6 ± 1.7 mV (G-V), and 3.2 ± 2.5 and 18.8 ± 2.1 mV (F-V; n = 3). The F-V relation was 41 mV left shifted from the G-V relation. (D and E) Representative ionic (D) and fluorescence (E) signals recorded from A397C in the presence of the ILT mutation during 100-ms voltage clamp pulses to +60 mV. The ILT mutation uncouples independent voltage sensor movement from the concerted opening transition (Smith-Maxwell et al., 1998a,b) resulting in a shift of the G-V relation to depolarized potentials (see F). (F) Mean G-V and F-V relations for Kv1.5 A397C ILT channels. V1/2 and k values for G-V and F-V relations were 131.0 ± 1.2 and 21.4 ± 0.8 mV (G-V), respectively, and −41.6 ± 1.8 and 20.6 ± 1.7 mV (F-V; n = 3). The F-V relation was therefore 173 mV left shifted from the G-V relation.
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fig7: Prevention of opening abolishes the dequenching of fluorescence. (A and B) Ionic (A) and fluorescence (B) signals recorded from TMRM attached at A397C in control conditions and in the presence of 10 mM 4-AP during 100-ms voltage clamp pulses from −80 to +60 mV. 10 mM 4-AP prevented channel opening and reduced current amplitude by 66 ± 6% (n = 3). (C) Mean G-V and F-V relations in the presence of 10 mM 4-AP. V1/2 and k values for G-V and F-V relations were 44.0 ± 2.8 and 23.6 ± 1.7 mV (G-V), and 3.2 ± 2.5 and 18.8 ± 2.1 mV (F-V; n = 3). The F-V relation was 41 mV left shifted from the G-V relation. (D and E) Representative ionic (D) and fluorescence (E) signals recorded from A397C in the presence of the ILT mutation during 100-ms voltage clamp pulses to +60 mV. The ILT mutation uncouples independent voltage sensor movement from the concerted opening transition (Smith-Maxwell et al., 1998a,b) resulting in a shift of the G-V relation to depolarized potentials (see F). (F) Mean G-V and F-V relations for Kv1.5 A397C ILT channels. V1/2 and k values for G-V and F-V relations were 131.0 ± 1.2 and 21.4 ± 0.8 mV (G-V), respectively, and −41.6 ± 1.8 and 20.6 ± 1.7 mV (F-V; n = 3). The F-V relation was therefore 173 mV left shifted from the G-V relation.

Mentions: As a further test of this hypothesis, we investigated the effects of preventing opening on the fluorescence report from Kv1.5 A397C channel. We measured the fluorescence report in the presence of 10 mM 4-AP or the ILT triple mutation (V407I, I410L, S414T), both of which dissociate voltage sensor movement from channel opening by stabilizing channels in preopen closed states (McCormack et al., 1994; Smith-Maxwell et al., 1998a,b; del Camino et al., 2005; Pathak et al., 2005). 10 mM 4-AP reduced the ionic current by about two thirds and abolished the rapid component of fluorescence (Fig. 7, A and B), reverting the fluorescence phenotype to that reported by Shaker A359C (compare the fluorescence trace in the presence of 4-AP with that in Fig. 2 B). In addition, 4-AP restored the left-shifted voltage dependence of the F-V relation (the V1/2 of the F-V was ∼41 mV left shifted from that of the G-V; Fig. 7 C). In a similar manner, at potentials (e.g., +60 mV) that stabilize ILT mutant channels in the activated-not-open state (del Camino et al., 2005), where little ionic current is observed (Fig. 7 D), the dequenching component of fluorescence from Kv1.5 ILT A397C channels was abolished and the Shaker-like monoexponential report of voltage sensor movement was rescued (Fig. 7 E). The F-V relation in ILT mutant channels was also left shifted from the G-V relation (the V1/2 of the F-V was −41.6 ± 1.8 mV; Fig. 7 F). These data clearly demonstrate that the dequenching component of fluorescence from A397C is associated with channel opening. Furthermore, these results show that the depolarized position of the F-V relation in Fig. 2 F does not reflect the true voltage dependence of voltage sensor movement, because it is influenced by an event that TMRM detects upon channel 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)

Prevention of opening abolishes the dequenching of fluorescence. (A and B) Ionic (A) and fluorescence (B) signals recorded from TMRM attached at A397C in control conditions and in the presence of 10 mM 4-AP during 100-ms voltage clamp pulses from −80 to +60 mV. 10 mM 4-AP prevented channel opening and reduced current amplitude by 66 ± 6% (n = 3). (C) Mean G-V and F-V relations in the presence of 10 mM 4-AP. V1/2 and k values for G-V and F-V relations were 44.0 ± 2.8 and 23.6 ± 1.7 mV (G-V), and 3.2 ± 2.5 and 18.8 ± 2.1 mV (F-V; n = 3). The F-V relation was 41 mV left shifted from the G-V relation. (D and E) Representative ionic (D) and fluorescence (E) signals recorded from A397C in the presence of the ILT mutation during 100-ms voltage clamp pulses to +60 mV. The ILT mutation uncouples independent voltage sensor movement from the concerted opening transition (Smith-Maxwell et al., 1998a,b) resulting in a shift of the G-V relation to depolarized potentials (see F). (F) Mean G-V and F-V relations for Kv1.5 A397C ILT channels. V1/2 and k values for G-V and F-V relations were 131.0 ± 1.2 and 21.4 ± 0.8 mV (G-V), respectively, and −41.6 ± 1.8 and 20.6 ± 1.7 mV (F-V; n = 3). The F-V relation was therefore 173 mV left shifted from the G-V relation.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Prevention of opening abolishes the dequenching of fluorescence. (A and B) Ionic (A) and fluorescence (B) signals recorded from TMRM attached at A397C in control conditions and in the presence of 10 mM 4-AP during 100-ms voltage clamp pulses from −80 to +60 mV. 10 mM 4-AP prevented channel opening and reduced current amplitude by 66 ± 6% (n = 3). (C) Mean G-V and F-V relations in the presence of 10 mM 4-AP. V1/2 and k values for G-V and F-V relations were 44.0 ± 2.8 and 23.6 ± 1.7 mV (G-V), and 3.2 ± 2.5 and 18.8 ± 2.1 mV (F-V; n = 3). The F-V relation was 41 mV left shifted from the G-V relation. (D and E) Representative ionic (D) and fluorescence (E) signals recorded from A397C in the presence of the ILT mutation during 100-ms voltage clamp pulses to +60 mV. The ILT mutation uncouples independent voltage sensor movement from the concerted opening transition (Smith-Maxwell et al., 1998a,b) resulting in a shift of the G-V relation to depolarized potentials (see F). (F) Mean G-V and F-V relations for Kv1.5 A397C ILT channels. V1/2 and k values for G-V and F-V relations were 131.0 ± 1.2 and 21.4 ± 0.8 mV (G-V), respectively, and −41.6 ± 1.8 and 20.6 ± 1.7 mV (F-V; n = 3). The F-V relation was therefore 173 mV left shifted from the G-V relation.
Mentions: As a further test of this hypothesis, we investigated the effects of preventing opening on the fluorescence report from Kv1.5 A397C channel. We measured the fluorescence report in the presence of 10 mM 4-AP or the ILT triple mutation (V407I, I410L, S414T), both of which dissociate voltage sensor movement from channel opening by stabilizing channels in preopen closed states (McCormack et al., 1994; Smith-Maxwell et al., 1998a,b; del Camino et al., 2005; Pathak et al., 2005). 10 mM 4-AP reduced the ionic current by about two thirds and abolished the rapid component of fluorescence (Fig. 7, A and B), reverting the fluorescence phenotype to that reported by Shaker A359C (compare the fluorescence trace in the presence of 4-AP with that in Fig. 2 B). In addition, 4-AP restored the left-shifted voltage dependence of the F-V relation (the V1/2 of the F-V was ∼41 mV left shifted from that of the G-V; Fig. 7 C). In a similar manner, at potentials (e.g., +60 mV) that stabilize ILT mutant channels in the activated-not-open state (del Camino et al., 2005), where little ionic current is observed (Fig. 7 D), the dequenching component of fluorescence from Kv1.5 ILT A397C channels was abolished and the Shaker-like monoexponential report of voltage sensor movement was rescued (Fig. 7 E). The F-V relation in ILT mutant channels was also left shifted from the G-V relation (the V1/2 of the F-V was −41.6 ± 1.8 mV; Fig. 7 F). These data clearly demonstrate that the dequenching component of fluorescence from A397C is associated with channel opening. Furthermore, these results show that the depolarized position of the F-V relation in Fig. 2 F does not reflect the true voltage dependence of voltage sensor movement, because it is influenced by an event that TMRM detects upon channel 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
Related in: MedlinePlus