Determining the relative ionic selectivity for the mHCN2-EGFP channel. Symmetrical solutions were used on both sides of the membrane. All recordings were collected in the presence of saturating concentration of cAMP (3 µM). (A) Current trace recorded with solutions containing 110 mM Na⁺ and 5 mM K⁺ as the charge carriers. (B) Current trace recorded with solutions containing 110 mM NH₄⁺ and 5 mM K⁺. (C) Cross-plots of fluorescence intensity versus current amplitude. Symmetric solutions contain 110 mM Na⁺ only. The slope was 0.676 a.u./pA. As the control, the slope of the condition with 110 mM K⁺ and 2 mM Na⁺ was 0.0067 a.u./pA. Thus, PK⁺/PNa⁺ based on the ratio of two slopes was 101 (in the absence of K⁺). Linear fit statistics: Pearson correlation coefficient, 0.766. (D) Results of 110 mM Na⁺ and 5 mM K⁺. The slope was 0.023 a.u./pA. PK⁺/PNa⁺ was 3.4 (in the presence of 5 mM K⁺). Linear fit statistics: Pearson correlation coefficient, 0.845. (E) Results of 110 mM NH₄⁺ only. The slope was 0.088 a.u./pA. PK⁺/PNH₄⁺ was 13.1 (in the absence of K⁺). Linear fit statistics: Pearson correlation coefficient, 0.811. (F) Results of 110 mM NH₄⁺ and 5 mM K⁺. The slope was 0.081 a.u./pA. PK⁺/PNH₄⁺ was 12.1 (in the presence of 5 mM K⁺). Linear fit statistics: Pearson correlation coefficient, 0.832. Green curves show the confidence bands (95%).