TMRM attached to residues in the S1–S2 linker of Kv1.2 report only slow changes in fluorescence emission in response to voltage. (A) An alignment of best fit for the amino acid residues of the S1–S2 linker region (underlined) of Shaker, Kv1.2 and Kv1.5. Residues in gray correspond to portions of the S1 and S2 helices. Residues tested for fluorescence are boxed, and those giving voltage-dependent deflections are in bold. (B and C) Representative fluorescence emissions recorded at 60 mV from I187C (B) and T219C (C) channels. (D) Mean G-V (closed symbols) and F-V (open symbols) relationships for I187C (diamonds) and T219C (triangles; n = 6–15) compared with Kv1.2 A291C fast and slow fluorescence components from Fig. 5 C (open circles and squares, respectively). Boltzmann fits to I187C data gave V_1/2 and k values of −50.6 ± 1.4 mV and 10.6 ± 0.5 mV for F-V and −17.7 ± 1.9 mV and 27.3 ± 1.0 mV for G-V. T219C half-activation potential and slope factor values were −15.5 ± 2.6 mV and 22.8 ± 0.3 mV and −70.8 ± 5.9 mV and 18.3 ± 2.0 mV for the G-V and F-V relationships. Error bars represent mean ± SEM.