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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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Other fluorophores report the unusual fluorescence signal. (A and B) Ionic current (A) and fluorescence (B) traces recorded from oocytes expressing Kv1.5 A397C channels labeled with PyMPO. Voltage clamp pulses were applied from −80 to +60 mV in 10-mV increments (100 ms duration) from a holding potential of −80 mV (only traces at −60, −30, 0, and +30 mV are shown). (C) Mean G-V and F-V relations for Kv1.5 A397C labeled with PyMPO (n = 4). Boltzmann fits of the data gave V1/2 and k values for G-V and F-V relations of 2.3 ± 1.9 and 17.8 ± 1.3 mV (G-V), respectively, and −6.5 ± 2.5 and 22.2 ± 1.7 mV (F-V), respectively. (D) Mean values for the time constants of the dequenching component of fluorescence measured from PyMPO attached to A397C and the time constants of the associated ionic current activation. The time constants show similar voltage dependence at test voltages ranging from +10 to +60 mV.
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fig5: Other fluorophores report the unusual fluorescence signal. (A and B) Ionic current (A) and fluorescence (B) traces recorded from oocytes expressing Kv1.5 A397C channels labeled with PyMPO. Voltage clamp pulses were applied from −80 to +60 mV in 10-mV increments (100 ms duration) from a holding potential of −80 mV (only traces at −60, −30, 0, and +30 mV are shown). (C) Mean G-V and F-V relations for Kv1.5 A397C labeled with PyMPO (n = 4). Boltzmann fits of the data gave V1/2 and k values for G-V and F-V relations of 2.3 ± 1.9 and 17.8 ± 1.3 mV (G-V), respectively, and −6.5 ± 2.5 and 22.2 ± 1.7 mV (F-V), respectively. (D) Mean values for the time constants of the dequenching component of fluorescence measured from PyMPO attached to A397C and the time constants of the associated ionic current activation. The time constants show similar voltage dependence at test voltages ranging from +10 to +60 mV.

Mentions: A similar fluorescence profile of channel gating was reported by another fluorophore, PyMPO (Fig. 5), that has different physical and spectral properties to TMRM. Ionic currents (Fig. 5 A) and the G-V relationship (Fig. 5 C), which had a V1/2 and k of 2.3 ± 1.9 and 17.8 ± 1.3 mV, respectively, were relatively unaffected by labeling with PyMPO. The fluorescence reports were very similar to those seen with TMRM, and included a transient component that appeared at around 0 mV, a potential at which significant numbers of channels opened (Fig. 5 B). It was noted that PyMPO fluorescence recordings bleached more rapidly during clamp pulses, and perfect baseline correction was not possible when full F-V relations were obtained. This explains the baseline drift seen in recordings at 0 and +30 mV in Fig. 5 B. With TMRM, the time constant of the fluorescence dequenching at +60 mV was 3.4 ± 0.6 ms and it represented 36 ± 3% of the total signal (Fig. 4; n = 4). The corresponding values with PyMPO were similar (Fig. 5 D); the time constant was 2.1 ± 0.5 ms and it represented 25 ± 8% of the total signal (n = 3), suggesting that the fluorescence changes observed are a faithful report of protein conformational changes. Upon repolarization, the return of both the TMRM and PyMPO fluorescence signals was rapid (see also Fig. 2 E), and showed no evidence of return of the rapid component of fluorescence. This suggests that the motions represented by the dequenching of fluorescence upon depolarization are slow to recover upon repolarization.


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)

Other fluorophores report the unusual fluorescence signal. (A and B) Ionic current (A) and fluorescence (B) traces recorded from oocytes expressing Kv1.5 A397C channels labeled with PyMPO. Voltage clamp pulses were applied from −80 to +60 mV in 10-mV increments (100 ms duration) from a holding potential of −80 mV (only traces at −60, −30, 0, and +30 mV are shown). (C) Mean G-V and F-V relations for Kv1.5 A397C labeled with PyMPO (n = 4). Boltzmann fits of the data gave V1/2 and k values for G-V and F-V relations of 2.3 ± 1.9 and 17.8 ± 1.3 mV (G-V), respectively, and −6.5 ± 2.5 and 22.2 ± 1.7 mV (F-V), respectively. (D) Mean values for the time constants of the dequenching component of fluorescence measured from PyMPO attached to A397C and the time constants of the associated ionic current activation. The time constants show similar voltage dependence at test voltages ranging from +10 to +60 mV.
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fig5: Other fluorophores report the unusual fluorescence signal. (A and B) Ionic current (A) and fluorescence (B) traces recorded from oocytes expressing Kv1.5 A397C channels labeled with PyMPO. Voltage clamp pulses were applied from −80 to +60 mV in 10-mV increments (100 ms duration) from a holding potential of −80 mV (only traces at −60, −30, 0, and +30 mV are shown). (C) Mean G-V and F-V relations for Kv1.5 A397C labeled with PyMPO (n = 4). Boltzmann fits of the data gave V1/2 and k values for G-V and F-V relations of 2.3 ± 1.9 and 17.8 ± 1.3 mV (G-V), respectively, and −6.5 ± 2.5 and 22.2 ± 1.7 mV (F-V), respectively. (D) Mean values for the time constants of the dequenching component of fluorescence measured from PyMPO attached to A397C and the time constants of the associated ionic current activation. The time constants show similar voltage dependence at test voltages ranging from +10 to +60 mV.
Mentions: A similar fluorescence profile of channel gating was reported by another fluorophore, PyMPO (Fig. 5), that has different physical and spectral properties to TMRM. Ionic currents (Fig. 5 A) and the G-V relationship (Fig. 5 C), which had a V1/2 and k of 2.3 ± 1.9 and 17.8 ± 1.3 mV, respectively, were relatively unaffected by labeling with PyMPO. The fluorescence reports were very similar to those seen with TMRM, and included a transient component that appeared at around 0 mV, a potential at which significant numbers of channels opened (Fig. 5 B). It was noted that PyMPO fluorescence recordings bleached more rapidly during clamp pulses, and perfect baseline correction was not possible when full F-V relations were obtained. This explains the baseline drift seen in recordings at 0 and +30 mV in Fig. 5 B. With TMRM, the time constant of the fluorescence dequenching at +60 mV was 3.4 ± 0.6 ms and it represented 36 ± 3% of the total signal (Fig. 4; n = 4). The corresponding values with PyMPO were similar (Fig. 5 D); the time constant was 2.1 ± 0.5 ms and it represented 25 ± 8% of the total signal (n = 3), suggesting that the fluorescence changes observed are a faithful report of protein conformational changes. Upon repolarization, the return of both the TMRM and PyMPO fluorescence signals was rapid (see also Fig. 2 E), and showed no evidence of return of the rapid component of fluorescence. This suggests that the motions represented by the dequenching of fluorescence upon depolarization are slow to recover upon repolarization.

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