Distinct voltage-dependent fluorescence changes by labeling spHCN ^R332C with ALEXA-488 or MTS-TAMRA (mixed isomer). (A) Chemical structures of the fluorophores used in this study. Arrows indicate site of thiol-maleimide conjugation (ALEXA-488) or S-S cleavage of MTS group (MTS-5(6)-TAMRA). (B) The predicted position of the labeling site R332 is shown in the cartoon rendering of two subunits of spHCN in the closed (depolarized) conformation with main functional components for one subunit highlighted (S1–3: blue; S4: red; S5: yellow; S6:green). CNBD, cyclic nucleotide binding domain. (C) Representative recordings of membrane current (I_m, upper panels) and simultaneous fluorescence (ΔF, lower panels) for ALEXA-488–labeled oocyte (left) and MTS-TAMRA–labeled oocyte (right) in response to the voltage step protocol shown. Oocytes were from the same donor frog recorded on consecutive days. Boxed regions expanded (×10) for voltage steps to −160 mV highlight the initial fast ΔF for ALEXA-488 labeling. The vertical dashed lines indicate the approximate end of the capacitive charging phase. ΔF shown as percentage of background fluorescence. Current and fluorescence were low-pass filtered at 400 Hz. (D) Parametric plots of ΔF and activating currents (I^act) for representative oocytes labeled with either ALEXA-488 (left panel) or MTS-TAMRA (right panel) for four hyperpolarizing potentials from −100 to −160 mV in 20-mV intervals. For the ALEXA-488 data, gray shading indicates region ∼60 ms after step onset, for which I^act and ΔF appear to correlate. For the MTS-TAMRA data, the initial 60 ms is shaded and expanded in C. (E) MTS-TAMRA data for the same oocyte as in B but using a 60-ms test pulse with the same potentials. Gray marked zone corresponds to same area in B (right panel). Superimposed lines highlight linear relation between early phase of ΔF and I^act for MTS-TAMRA labeling for −100 and −160 mV.