K470 is required for the interaction of Kv2.1 with SUMO or Ubc9. CFP-Kv2.1 and CFP-Kv2.1-K470Q channels were studied with YFP fused to Kv2.1-K470Q, SUMO1₁₀₁, or Ubc9. Data are the mean time constant for CFP decay ± SEM for two to five areas of five to seven cells with continuous excitation. (A) CHO cells expressing CFP-Kv2.1 or CFP-Kv2.1-K470Q. Bar, 10 µm. (B) Exponential decay curves for CFP-Kv2.1 coexpressed with YFP-SUMO1₁₀₁ (○) or free YFP (•). The decrease in normalized fluorescence intensity was fit by a single exponential to give the decay time constant (τ). (C) FRET was observed (mean τ ± SEM) between CFP-Kv2.1 (dark gray) and YFP-SUMO1₁₀₁ (Su₁₀₁) when K470 was present and SUMO1 was competent to form isopeptide bonds. The mean τ for CFP-Kv2.1 decay was increased upon expression with YFP-SUMO1₁₀₁ (24.8 ± 2.4 s), but not upon expression with YFP-SUMO1₉₅ (Su₉₅, τ = 8.9 ± 1.3 s) compared with free YFP (Free; τ = 9 ± 1.1 s). Increased mean τ ± SEM upon expression of CFP-Kv2.1-K470Q (white) and YFP-Kv2.1-K470Q reflects channels with both subunits (τ = 26.2 ± 2.9 s). Unlike CFP-Kv2.1, the mean τ of CFP-Kv2.1-K470Q was not significantly altered by coexpression with YFP-SUMO1₁₀₁ (τ = 10 ± 3.5 s), YFP-SUMO1₉₅ (τ = 10.2 ± 1.8 s), or free YFP (τ = 9.2 ± 1.0 s). The interaction of CFP-Kv2.1 with YFP-Ubc9 requires K470; thus, the mean τ was greater for CFP-Kv2.1 (dark gray; τ = 23.2 ± 2.4 s) than for CFP-Kv2.1-K470Q (white; τ = 10 ± 2.9 s).