Two SUMO1 monomers associate with Kv2.1 channels to yield a maximal effect. GFP-tagged subunits were studied at the CHO cell surface by TIRF and single-particle photobleaching. Kv2.1 and Kv2.1-K470Q were expressed as monomer, dimer, or tetrameric constructs. G-V relationships for the channels were determined in whole cell mode with control buffer, 250 pM SENP1, or 75 pM SUMO1₁₀₁ in the pipette, as in Fig. 4. (A; left). Time course of photobleaching for a representative GFP-Kv2.1 fluorescent particle showing four steps as expected for a tetrameric channel. (Inset) Fluorescent particles in a cell expressing GFP-Kv2.1. Bar, 60 pixels. (Right) Histogram showing 117 GFP-Kv2.1 particles (gray bars), of which 9% had four bleaching stops, 42% had three, 14% had two, and 5% had a single step. The distribution was similar for 123 GFP-Kv2.1-K470Q particles (white bars), with four, three, two, and one steps at 38, 41, 15, and 6%, respectively. (B; left) Photobleaching time course for a representative GFP-SUMO1₁₀₁ fluorescent Kv2.1 particle showing two bleaching steps with continuous excitation. (Right) Histogram for GFP-SUMO1₁₀₁ expressed with untagged Kv2.1 channels showing that 75% of 80 fluorescent particles had two bleaching steps. No particles with three or more bleaching steps were observed. No discrete immobile particles were observed when GFP-SUMO1₁₀₁ was coexpressed with Kv2.1-K470Q channels. (C) Lys-Gln (gray-white) or Gln-Lys (white-gray) tandem constructs and Lys-Gln-Lys-Gln or Gln-Lys-Lys-Gln tetramer constructs were expressed with GFP-SUMO1₁₀₁. These channels with only two K470 sites showed no more than two bleaching steps. Two steps were recorded in 72% of 50 spots with Lys-Gln channels and 73% of 58 spots with Gln-Lys channels; similar to channels of wild-type monomers, Lys-Gln or Gln-Lys channels showed shifts in V_1/2 of 34 ± 4 and 35 ± 3 mV (with no change in V_s) between SENP1 and SUMO1₁₀₁ treatment. Similarly, two steps were recorded in 71% of 49 spots for Lys-Gln-Lys-Gln channels and a 33 ± 4–mV shift in V_1/2 with no change in V_s was observed between SENP1 and SUMO1₁₀₁ exposure. (D) All 49 spots studied with Gln-Lys-Lys-Gln channels and GFP-SUMO1₁₀₁ bleached in a single step. Here, the shift in V_1/2 between SENP1 and SUMO1₁₀₁ treatment was only 13 ± 8 mV, with no change in V_s. One bleaching step was also observed in all 69 Lys-Gln-Gln-Gln channels and all 58 Gln-Lys-Gln-Gln channels. The total shift in V_1/2 between SENP1 and SUMO1₁₀₁ treatment for these channels was 15 ± 3 and 14 ± 3 mV, respectively.