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 and 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 ( = 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 ( = 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 ( = 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 ( = 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 ( = 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 ( = 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, and values were 5.8 ± 1.8 and 10.9 ± 1.5 mV ( = 4), respectively, and the corresponding values for A397C were 31.0 ± 2.4 and 15.5 ± 1.9 mV ( = 4).Copyright information:Taken from "Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel"The Journal of General Physiology 2008;132(2):209-222.Published online Jan 2008PMCID:PMC2483330.