Vaid M , Claydon TW , Rezazadeh S , Fedida D .

	Figure 1. TMRM fluorescence from Kv1.5 WT and Kv1.5 C268V channels. (A, B, D, and E) Ionic current (A and D) and fluorescence (B and E) traces recorded from oocytes expressing Kv1.5 WT (A and B) or Kv1.5 C268V (D and E) channels labeled with TMRM. 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 +60 mV traces are shown for clarity). (C and F) Mean G-V relations for Kv1.5 WT (C; n = 9) and Kv1.5 C268V (F; n = 4). V1/2 and k values for the G-V relation of Kv1.5 channels were 1.2 ± 1.3 and 12.5 ± 1.0 mV, respectively. The V1/2 and k values for the G-V relations of Kv1.5 C268V were −5.7 ± 1.0 and 10.8 ± 0.8 mV, respectively. The V1/2 values for the G-V relations of Kv1.5 WT and Kv1.5 C268V are significantly different P < 0.01 (unpaired t test). Note that error bars generally fall within the plotted points in C and F.
	Figure 2. Characteristics of the TMRM fluorescence report from Kv1.5 A397C channels. (A, B, D, and E) Representative ionic current (A and D) and fluorescence (B and E) traces recorded from oocytes expressing Shaker A359C (A and B) or Kv1.5 A397C (D and E) channels labeled with TMRM. 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 highlighted traces are shown for clarity). (C and F) Mean G-V and F-V relations for Shaker A359C (C; n = 3) and Kv1.5 A397C (F; n = 4). V1/2 and k values for the G-V relation of Shaker A359C channels were −16.2 ± 1.3 and 16.8 ± 1.1 mV, respectively, and the corresponding values for the F-V relation were −40.9 ± 1.9 and 17.0 ± 1.7 mV, respectively. V1/2 and k values for the G-V relation of Kv1.5 A397C were 7.3 ± 1.6 and 16.4 ± 1.2 mV, respectively, and the corresponding values for the F-Vpeak relation were 1.9 ± 1.9 and 19.0 ± 1.3 mV, respectively. The F-Vpeak relation was not significantly shifted from the G-V relation. The voltage dependence of the dequenching component of fluorescence from Kv1.5 A397C (calculated as the peak minus end fluorescence amplitude, F-Vdecay) is also shown in F; V1/2 and k values were 31.0 ± 2.4 and 15.5 ± 1.9 mV, respectively.
	Figure 3. Sites facing the pore report the rapid dequenching of fluorescence. (A and B) Representative ionic and fluorescence signals recorded from TMRM attached at each site in the Kv1.5 S3–S4 linker from M394C to V401C during 100 ms voltage clamp pulses from −80 to +60 mV. Scale bars represent 30 μA currents (A) and 1% ΔF/F fluorescence deflections (B), respectively. (C) Mean G-V (•) and F-V (○) relations for each mutation. Boltzmann fits of the data gave V1/2 and k values for G-V and F-V relations of 6.9 ± 1.9 and 17.3 ± 1.4 mV (G-V), respectively, and −10.5 ± 1.9 and 19.4 ± 1.4 mV (F-V), respectively, for M394C (n = 4); 9.0 ± 1.4 and 17.2 ± 1.0 mV (G-V), respectively, and −54.7 ± 0.8 and 9.7 ± 0.7 mV (F-V), respectively, for S395C (n = 7); −19.7 ± 1.8 and 16.2 ± 1.5 mV (G-V), respectively, and −31.5 ± 3.3 and 25.1 ± 2.8 mV (F-V), respectively, for L396C (n = 3); 7.4 ± 1.6 and 16.4 ± 1.2 mV (G-V), respectively, and 2.0 ± 1.9 and 18.9 ± 1.3 mV (F-V), respectively, for A397C (n = 4); −18.1 ± 3.4 and 21.6 ± 2.7 mV (G-V), respectively, and −66.4 ± 1.6 and 7.2 ± 1.5 mV (F-V), respectively, for L399C (n = 3); 10.8 ± 1.2 and 13.3 ± 0.9 mV (G-V), respectively, and 25.7 ± 2.2 and 23.3 ± 1.0 mV (F-V), respectively, for V401C (n = 7); voltage-dependent fluorescence deflections were not evident with TMRM attached at I398C, and R400C channels did not express ionic current. The voltage dependence of the dequenching component of fluorescence (calculated as the peak minus end fluorescence amplitude) is also shown (▾) for M394C and A397C. In the case of M394C, V1/2 and k values were 5.8 ± 1.8 and 10.9 ± 1.5 mV (n = 4), respectively, and the corresponding values for A397C were 31.0 ± 2.4 and 15.5 ± 1.9 mV (n = 4).
	Figure 4. 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.
	Figure 5. 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.
	Figure 6. 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).
	Figure 7. 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.
	Figure 8. Time course for the recovery of the de-quenching component of fluorescence. (A and B) Ionic current and fluorescence signals recorded during the voltage protocol shown. The time course of the recovery of the dequenching component fluorescence amplitude was assessed during 75-ms test pulses to +60 mV applied at different intervals following a 15-ms conditioning pulse to +60 mV. The fluorescence signal, but not ionic current during the test pulse has been truncated for purposes of clarity. The dashed lines mark the conditioning pulse instantaneous ion current (in A) or fluorescence amplitude (in B) for comparison. (C) Fractional recovery of the dequenching component of fluorescence amplitude (filled symbols) or the recovery of instantaneous current (open symbols) recorded from three different oocytes during test pulses applied at different intervals following 15-ms conditioning pulses. Data are shown as mean ± SEM. When fitted with a single exponential function, the instantaneous current recovery had a time constant of 10.7 ± 0.8 ms and that of fluorescence was significantly slower 20.7 ± 3.7 ms (P < 0.05, n = 3).
	Figure 9. Effect of prepulse duration on the time course of fluorescence recovery. (A and B) Ionic current and fluorescence signals recorded from TMRM-labeled Kv1.5 A397C channels during a 75-ms test pulse to +60 mV applied 7.5 ms and 37.5 ms following a +60-mV conditioning pulse of either 15 ms (A) or 5 ms (B) duration. The dotted line marks the conditioning pulse peak fluorescence amplitude for comparison.
	Figure 10. Immobilization of the selectivity filter gate abolishes the fluorescence dequenching. (A and B) Ionic current and fluorescence signals recorded from Kv1.5 A397C channels during 100-ms pulses to +60 mV in the presence of 3 and 99 mM external K+. (C) Mean F-V relation with 99 mM K+ plotted alongside the G-V and F-V relations obtained with 3 mM K+ to demonstrate the voltage dependence of voltage sensor movement when the selectivity filter gate is immobilized by high external K+. The G-V relation with 99 mM K+ is not shown because of the large error associated with calculations around the reversal potential (∼0 mV). V1/2 and k values for the F-V relation with 99 mM K+ were −36.4 ± 1.2 and 12.8 ± 1.0 mV, respectively (n = 3). V1/2 and k values for the G-V and F-V relations with 3 mM K+ were 8.1 ± 2.5 and 18.3 ± 1.7 mV (G-V) and 4.3 ± 2.0 and 21.5 ± 1.3 mV (F-V), respectively (n = 3). The F-V relation with high external K+ was therefore 40 mV left shifted from the G-V relation. (D) Ionic current and fluorescence signals from Kv1.5 A397C W472F mutant channels (W472F is equivalent to the W434F mutation in Shaker channels) during a 100-ms voltage clamp pulse from −80 to +60 mV. Note that only small leak currents were observed from oocytes injected with Kv1.5 A397C W472F. Similar recordings were obtained from nine (W472F) other cells.
	Figure 11. 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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