Comparison of voltage-dependent properties of unlabeled and labeled spHCN ^R332C . (A) Conductance (G) derived from tail currents and total ΔF (∆F_total) measured at the end of 1-s test pulse normalized to respective maxima predicted from Boltzmann fit to data sets from individual oocytes and then pooled (see Materials and methods). Open symbols: G; filled symbols, ∆F_total. Unlabeled spHCN^R332C: n = 3; ALEXA-488–labeled spHCN^R332C: n = 5; MTS-TAMRA labeled; n = 5. Symbols shown in boxed inset. (B) Activation time constants plotted as a function of test pulse for current (τ^I) and ΔF (τ^F) compared for labeled and unlabeled cells. For both labels, activation currents were fit with two exponential: for ALEXA-488–labeled oocytes, ΔF was fit with a single rising exponential with variable offset for ALEXA-488 labeling and a double exponential for MTS-TAMRA labeling. ALEXA-488–labeled spHCN^R332C: n = 15; MTS-TAMRA–labeled spHCN^R332C: n = 9; unlabeled spHCN^R332C: n = 6. Symbols shown in boxed inset. A one-way ANOVA test reported no significant difference (P < 0.05) between the means of the fast component of τ^I for the unlabeled and each labeled case in the potential range −160 to −80 mV. There was no significant difference (t test, P < 0.05) between the means in the potential range −160 to −80 mV when comparing the fast components of τ^I and τ^F for TAMRA labeling reported. (C) For ALEXA-488–labeled oocytes, ∆F_total was resolved into two components (∆F_fast, ∆F_slow) when fit with a single rising exponential with variable offset, where ∆F_fast represents the fit offset and ∆F_slow represents the amplitude of the exponential component. Same data set as in B. Data shown normalized to ∆F_total. Symbols shown in boxed inset. (D) For MTS-TAMRA–labeled oocytes, ∆F_totalwas resolved into two components (∆F_fast, ∆F_slow) when fit with a double exponential function_. Same data set as in B. Data shown normalized to ∆F_total. Symbols shown in boxed inset.