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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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Voltage dependence of the dequenching of fluorescence and ionic current activation. (A) Mean values for the time constants of the dequenching component of fluorescence measured from TMRM attached to either M394C or A397C and the time constants of the associated ionic current activation, measured between +10 and +60 mV. (B) Relative amplitude of the dequenching component of fluorescence, normalized to the total fluorescence deflection on depolarization, measured from TMRM attached at either M394C or A397C.
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fig4: Voltage dependence of the dequenching of fluorescence and ionic current activation. (A) Mean values for the time constants of the dequenching component of fluorescence measured from TMRM attached to either M394C or A397C and the time constants of the associated ionic current activation, measured between +10 and +60 mV. (B) Relative amplitude of the dequenching component of fluorescence, normalized to the total fluorescence deflection on depolarization, measured from TMRM attached at either M394C or A397C.

Mentions: The different report of fluorophore environmental change observed in Kv1.5 (Fig. 2, D–F) may suggest that very different voltage sensor conformational changes occur in these channels than in Shaker channels. However, a fluorescence scan of the S3–S4 linker of Kv1.5 (Fig. 3) reveals that the complex structural changes are only reported by TMRM at two sites, A397C and M394C. This cannot be accounted for by differences in ionic current waveforms (Fig. 3 A) or changes in the G-V relationships with the different linker mutants (Fig. 3 C), since these all appear similar. Both A397C and M394C report a voltage-dependent acceleration of the dequenching component and an increase in its contribution to the total fluorescence change upon depolarization (Fig. 4). Other sites in the scan do not report the rapid component of fluorescence and produce deflections that are, in general, similar to those described from the equivalent sites in Shaker channels (Gandhi et al., 2000; Pathak et al., 2007). This suggests that Kv1.5 voltage sensor movement per se is not dissimilar to that of Shaker channels. Furthermore, given that the functional data suggests that the C-terminal end of the S3–S4 linker likely adopts an α-helical structure, placing M394C and A397C on the same side of the helix, (Li-Smerin et al., 2000; Gonzalez et al., 2001; Li-Smerin and Swartz, 2001), and at least M394C and A397C sense the same microenvironment changes, these data suggest that the fluorescence changes represent local protein rearrangements rather than global reconfigurations. Since M394C and A397C come close to the pore domain upon depolarization (Gandhi et al., 2000; Elinder et al., 2001), we hypothesized that TMRM attached at M394C or A397C might be reporting on an additional structural rearrangement that is associated with opening of the pore. In support of this, the time constant data in Fig. 4 A show a reasonably good match between the time constants of channel activation (filled symbols) and the fast component of fluorescence dequenching, especially for M394C (open triangles).


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)

Voltage dependence of the dequenching of fluorescence and ionic current activation. (A) Mean values for the time constants of the dequenching component of fluorescence measured from TMRM attached to either M394C or A397C and the time constants of the associated ionic current activation, measured between +10 and +60 mV. (B) Relative amplitude of the dequenching component of fluorescence, normalized to the total fluorescence deflection on depolarization, measured from TMRM attached at either M394C or A397C.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Voltage dependence of the dequenching of fluorescence and ionic current activation. (A) Mean values for the time constants of the dequenching component of fluorescence measured from TMRM attached to either M394C or A397C and the time constants of the associated ionic current activation, measured between +10 and +60 mV. (B) Relative amplitude of the dequenching component of fluorescence, normalized to the total fluorescence deflection on depolarization, measured from TMRM attached at either M394C or A397C.
Mentions: The different report of fluorophore environmental change observed in Kv1.5 (Fig. 2, D–F) may suggest that very different voltage sensor conformational changes occur in these channels than in Shaker channels. However, a fluorescence scan of the S3–S4 linker of Kv1.5 (Fig. 3) reveals that the complex structural changes are only reported by TMRM at two sites, A397C and M394C. This cannot be accounted for by differences in ionic current waveforms (Fig. 3 A) or changes in the G-V relationships with the different linker mutants (Fig. 3 C), since these all appear similar. Both A397C and M394C report a voltage-dependent acceleration of the dequenching component and an increase in its contribution to the total fluorescence change upon depolarization (Fig. 4). Other sites in the scan do not report the rapid component of fluorescence and produce deflections that are, in general, similar to those described from the equivalent sites in Shaker channels (Gandhi et al., 2000; Pathak et al., 2007). This suggests that Kv1.5 voltage sensor movement per se is not dissimilar to that of Shaker channels. Furthermore, given that the functional data suggests that the C-terminal end of the S3–S4 linker likely adopts an α-helical structure, placing M394C and A397C on the same side of the helix, (Li-Smerin et al., 2000; Gonzalez et al., 2001; Li-Smerin and Swartz, 2001), and at least M394C and A397C sense the same microenvironment changes, these data suggest that the fluorescence changes represent local protein rearrangements rather than global reconfigurations. Since M394C and A397C come close to the pore domain upon depolarization (Gandhi et al., 2000; Elinder et al., 2001), we hypothesized that TMRM attached at M394C or A397C might be reporting on an additional structural rearrangement that is associated with opening of the pore. In support of this, the time constant data in Fig. 4 A show a reasonably good match between the time constants of channel activation (filled symbols) and the fast component of fluorescence dequenching, especially for M394C (open triangles).

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