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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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The dequenching of fluorescence is associated with late transitions in the activation pathway. (A) Ionic currents and fluorescence signals recorded from TMRM attached at A397C during a 100-ms test pulse to +60 mV following a 100-ms conditioning pulse to either −120 or −40 mV. The dequenching component of fluorescence remains robust in channels that activate from preactivated states, suggesting that the dequenching component does not reflect voltage sensor transitions early in the activation pathway. (B) Mean values for the time constant and relative amplitude of the dequenching component of fluorescence during a test pulse to +60 mV from a range of prepulse potentials (n = 3).
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fig6: The dequenching of fluorescence is associated with late transitions in the activation pathway. (A) Ionic currents and fluorescence signals recorded from TMRM attached at A397C during a 100-ms test pulse to +60 mV following a 100-ms conditioning pulse to either −120 or −40 mV. The dequenching component of fluorescence remains robust in channels that activate from preactivated states, suggesting that the dequenching component does not reflect voltage sensor transitions early in the activation pathway. (B) Mean values for the time constant and relative amplitude of the dequenching component of fluorescence during a test pulse to +60 mV from a range of prepulse potentials (n = 3).

Mentions: As a first test of this hypothesis, we compared the effect of the holding potential on the fluorescence report from Kv1.5 A397C channels (Fig. 6). If the transient dequenching component of fluorescence is associated with late transitions to the open state rather than voltage sensor transitions early in the activation pathway, the fluorescence report from channels that open from hyperpolarized potentials (i.e., −120 mV) should be the same as that from channels opening from preactivated states (i.e., −40 mV). Data in Fig. 6 A shows ionic currents and fluorescence signals from TMRM attached at A397C recorded during a test pulse applied immediately after a conditioning pulse to either −120 or −40 mV. The ionic current records show that channel opening was more rapid following a conditioning pulse to −40 mV, which collects channels in preactivated states, than following a pulse to −120 mV, which collects channels in early closed states. Consistent with this, the fluorescence signal recorded following a prepulse to −40 mV dequenched more rapidly than following a pulse to −120 mV. However, the unique fluorescence profile that we observed in Kv1.5 channels was maintained following a prepulse to −40 mV, and the relative amplitude of the rapid fluorescence dequenching was unchanged by alterations in the holding potential (Fig. 6 B).


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)

The dequenching of fluorescence is associated with late transitions in the activation pathway. (A) Ionic currents and fluorescence signals recorded from TMRM attached at A397C during a 100-ms test pulse to +60 mV following a 100-ms conditioning pulse to either −120 or −40 mV. The dequenching component of fluorescence remains robust in channels that activate from preactivated states, suggesting that the dequenching component does not reflect voltage sensor transitions early in the activation pathway. (B) Mean values for the time constant and relative amplitude of the dequenching component of fluorescence during a test pulse to +60 mV from a range of prepulse potentials (n = 3).
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fig6: The dequenching of fluorescence is associated with late transitions in the activation pathway. (A) Ionic currents and fluorescence signals recorded from TMRM attached at A397C during a 100-ms test pulse to +60 mV following a 100-ms conditioning pulse to either −120 or −40 mV. The dequenching component of fluorescence remains robust in channels that activate from preactivated states, suggesting that the dequenching component does not reflect voltage sensor transitions early in the activation pathway. (B) Mean values for the time constant and relative amplitude of the dequenching component of fluorescence during a test pulse to +60 mV from a range of prepulse potentials (n = 3).
Mentions: As a first test of this hypothesis, we compared the effect of the holding potential on the fluorescence report from Kv1.5 A397C channels (Fig. 6). If the transient dequenching component of fluorescence is associated with late transitions to the open state rather than voltage sensor transitions early in the activation pathway, the fluorescence report from channels that open from hyperpolarized potentials (i.e., −120 mV) should be the same as that from channels opening from preactivated states (i.e., −40 mV). Data in Fig. 6 A shows ionic currents and fluorescence signals from TMRM attached at A397C recorded during a test pulse applied immediately after a conditioning pulse to either −120 or −40 mV. The ionic current records show that channel opening was more rapid following a conditioning pulse to −40 mV, which collects channels in preactivated states, than following a pulse to −120 mV, which collects channels in early closed states. Consistent with this, the fluorescence signal recorded following a prepulse to −40 mV dequenched more rapidly than following a pulse to −120 mV. However, the unique fluorescence profile that we observed in Kv1.5 channels was maintained following a prepulse to −40 mV, and the relative amplitude of the rapid fluorescence dequenching was unchanged by alterations in the holding potential (Fig. 6 B).

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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