BACKGROUND hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. RESULTS Tetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1-S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V((1/2)) of activation to -27.5+/-2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1-S2 linker cysteines with valines allowed unobstructed recording of S3-S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-V(ON), with V((1/2)) (,1) = -37.8+/-1.7 mV, and V((1/2)) (,2) = 43.5+/-7.9 mV. The first phase, V((1/2)) (,1), was approximately 20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V((1/2)) = -18.3+/-1.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V((1/2)) = -34.4+/-1.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V((1/2)) = -20.6+/-1.2, k = 11.4 mV) and L520C quenching during depolarization (V((1/2)) = -26.8+/-1.0, k = 13.3 mV) matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from -40 to -110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing. CONCLUSIONS THE DATA INDICATE: 1) that rapid environmental changes occur at the outer end of S4 in hERG channels that underlie channel activation gating, and 2) that secondary slower changes reflect channel pore opening during sustained depolarizations, and channel closing upon repolarization. 3) No direct evidence was obtained of conformational changes related to inactivation from fluorophores attached at the outer end of S4.