Figure 4.
Figure 4. SUMO1 regulates the voltage dependence of Kv2.1 via K470. Whole cell recording with solution A in the bath and solution B in the pipette (Table I); macropatch electrodes were filled with solution A, and the perfusate was solution B with peptides. (A) Kv2.1 or Kv2.1-K470Q channels studied in CHO cells in whole cell mode under control conditions (■; n = 15 cells), with 75 pM SUMO1101 (•; n = 12), or with 250 pM SENP1 in the pipette (▴; n = 12) with 500-ms test pulses to 50 from −80 mV every 10 s. (Left) Current traces. (Middle) Mean I-V relationships. (Right) Normalized G-V relationships. Biophysical parameters are summarized in Table I. Bars are 2 nA and 200 ms. Standard error bars are within symbols where not visible. Wild-type Kv2.1 channels showed a 36 ± 6–mV shift between exposure to SUMO1101 and SENP1, whereas K470Q channels were insensitive to both reagents. (B) Kv2.1 or Kv2.1-K470Q channels expressed in hippocampal neurons and studied as in Fig. 1, with 5 mM TEA in the bath. (Top) Neurons with Kv2.1 channels show increased activity with 7 pM SUMO1101 and 75 pM SUMO1101. (Bottom) Neurons with Kv2.1-K470Q channels were insensitive to 7 pM SUMO1101 and 75 pM SUMO1101. Values for Vm, RIN, and Ff are in Table S3. (C) Kv2.1 or Kv2.1-K470Q channels studied in CHO cells in excised plasma membrane patches. (Top left) Wild-type Kv2.1 channel currents (n = 6) increased from a mean starting value of 104 ± 11 to 150 ± 19 pA upon the application of 250 pM SENP1 (first arrow), with a half-maximal effect in 6 ± 2 s. Subsequent application of 75 pM SUMO1101 (second arrow) suppressed current to 74 ± 9 pA, with a half-maximal effect in 8 ± 3 s. The reapplication of SENP1 (third arrow) restored current to 147 ± 14 pA, a level that was stable despite washout of SENP1 (WASH). Half-block of Kv2.1 in excised patches by 15 mM TEA in the bath was 1.2 ± 0.8 s (n = 4 cells). (Bottom left) Kv2.1-K470Q channels (n = 4) had a mean starting value of 111 ± 9 pA and were not altered during the five-phase regimen in the top panel. (Top right) Wild-type Kv2.1 channel currents (n = 4) are suppressed (mean starting value, 140 ± 21 pA/pF; n = 4 patches) by 75 pM SUMO1101, and the effect is stable on the washout of SUMO1 peptide (WASH). (Bottom right) SUMO1101 had no effect on Kv2.1-K470Q channel currents (mean starting value, 124 ± 13 pA/pF; n = 4).

SUMO1 regulates the voltage dependence of Kv2.1 via K470. Whole cell recording with solution A in the bath and solution B in the pipette (Table I); macropatch electrodes were filled with solution A, and the perfusate was solution B with peptides. (A) Kv2.1 or Kv2.1-K470Q channels studied in CHO cells in whole cell mode under control conditions (■; n = 15 cells), with 75 pM SUMO1101 (•; n = 12), or with 250 pM SENP1 in the pipette (▴; n = 12) with 500-ms test pulses to 50 from −80 mV every 10 s. (Left) Current traces. (Middle) Mean I-V relationships. (Right) Normalized G-V relationships. Biophysical parameters are summarized in Table I. Bars are 2 nA and 200 ms. Standard error bars are within symbols where not visible. Wild-type Kv2.1 channels showed a 36 ± 6–mV shift between exposure to SUMO1101 and SENP1, whereas K470Q channels were insensitive to both reagents. (B) Kv2.1 or Kv2.1-K470Q channels expressed in hippocampal neurons and studied as in Fig. 1, with 5 mM TEA in the bath. (Top) Neurons with Kv2.1 channels show increased activity with 7 pM SUMO1101 and 75 pM SUMO1101. (Bottom) Neurons with Kv2.1-K470Q channels were insensitive to 7 pM SUMO1101 and 75 pM SUMO1101. Values for Vm, RIN, and Ff are in Table S3. (C) Kv2.1 or Kv2.1-K470Q channels studied in CHO cells in excised plasma membrane patches. (Top left) Wild-type Kv2.1 channel currents (n = 6) increased from a mean starting value of 104 ± 11 to 150 ± 19 pA upon the application of 250 pM SENP1 (first arrow), with a half-maximal effect in 6 ± 2 s. Subsequent application of 75 pM SUMO1101 (second arrow) suppressed current to 74 ± 9 pA, with a half-maximal effect in 8 ± 3 s. The reapplication of SENP1 (third arrow) restored current to 147 ± 14 pA, a level that was stable despite washout of SENP1 (WASH). Half-block of Kv2.1 in excised patches by 15 mM TEA in the bath was 1.2 ± 0.8 s (n = 4 cells). (Bottom left) Kv2.1-K470Q channels (n = 4) had a mean starting value of 111 ± 9 pA and were not altered during the five-phase regimen in the top panel. (Top right) Wild-type Kv2.1 channel currents (n = 4) are suppressed (mean starting value, 140 ± 21 pA/pF; n = 4 patches) by 75 pM SUMO1101, and the effect is stable on the washout of SUMO1 peptide (WASH). (Bottom right) SUMO1101 had no effect on Kv2.1-K470Q channel currents (mean starting value, 124 ± 13 pA/pF; n = 4).

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