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Table I.

Biophysical parameters for hippocampal neurons, native IDR currents, and cloned Kv2.1 channels

Treatment  Hippocampal neurons Native IDR Cloned Kv2.1 Cloned K470Q 
 Dose Vm Rin Ff V1/2 Vs IPEAK V1/2 Vs IPEAK V1/2 Vs IPEAK 
 pM mV  Hz mV  pA/pF mV  pA/pF mV  pA/pF 
None  −58 ± 4 65 ± 4 5 ± 2 22 ± 2 14 ± 3 72 ± 12 21 ± 4 9 ± 1 245 ± 16 3 ± 3 13 ± 3 382 ± 24 
SUMO292 75 −50 ± 4a 87 ± 5a 15 ± 5a 40 ± 3b 13 ± 2 20 ± 7b 36 ± 4b 10 ± 2 62 ± 10b 4 ± 4 11 ± 6 374 ± 21 
SUMO392 75 −48 ± 6a 86 ± 5a 14 ± 4a 39 ± 4b 12 ± 4 21 ± 7b 36 ± 6b 9 ± 3 62 ± 11b 4 ± 4 11 ± 7 373 ± 20 
SUMO1101 −52 ± 3 73 ± 6 8 ± 2 28 ± 3a 12 ± 3 35 ± 8b 28 ± 3a 12 ± 3 100 ± 15b 4 ± 8 13 ± 4 378 ± 17 
SUMO1101 75 −49 ± 4a 84 ± 5a 13 ± 3a 39 ± 3b 12 ± 3 22 ± 8b 37 ± 4b 10 ± 2 61 ± 8b 4 ± 3 11 ± 4 375 ± 19 
SUMO1101 250 −47 ± 6a 86 ± 5a 14 ± 4a 37 ± 4b 11 ± 4 21 ± 9b 35 ± 6b 9 ± 4 63 ± 9b 3 ± 4 12 ± 6 370 ± 24 
SUMO195 75 −60 ± 5 63 ± 6 6 ± 4 20 ± 3 13 ± 3 73 ± 7 20 ± 3 13 ± 3 249 ± 11 5 ± 4 13 ± 3 372 ± 15 
denatured SUMO1101 75 −57 ± 5 64 ± 3 5 ± 2 19 ± 4 12 ± 3 70 ± 8 20 ± 5 11 ± 4 240 ± 20 4 ± 4 13 ± 3 378 ± 18 
SENP1 250 −65 ± 5a 51 ± 5a 1 ± 2a 3 ± 4b 13 ± 4 100 ± 18b 1 ± 4b 10 ± 2 370 ± 23b 4 ± 4 13 ± 4 366 ± 21 
SENP1-C603S 250 −57 ± 5 64 ± 6 6 ± 3 20 ± 4 13 ± 4 72 ± 14 19 ± 6 9 ± 3 249 ± 17 4 ± 3 13 ± 3 388 ± 21 
Δ SENP-SUMO  16 ± 6 33 ± 8 12 ± 4 36 ± 5 0 ± 5 78 ± 20 36 ± 6 0 ± 3 309 ± 24 0 ± 5 0 ± 6 9 ± 28 
Treatment  Hippocampal neurons Native IDR Cloned Kv2.1 Cloned K470Q 
 Dose Vm Rin Ff V1/2 Vs IPEAK V1/2 Vs IPEAK V1/2 Vs IPEAK 
 pM mV  Hz mV  pA/pF mV  pA/pF mV  pA/pF 
None  −58 ± 4 65 ± 4 5 ± 2 22 ± 2 14 ± 3 72 ± 12 21 ± 4 9 ± 1 245 ± 16 3 ± 3 13 ± 3 382 ± 24 
SUMO292 75 −50 ± 4a 87 ± 5a 15 ± 5a 40 ± 3b 13 ± 2 20 ± 7b 36 ± 4b 10 ± 2 62 ± 10b 4 ± 4 11 ± 6 374 ± 21 
SUMO392 75 −48 ± 6a 86 ± 5a 14 ± 4a 39 ± 4b 12 ± 4 21 ± 7b 36 ± 6b 9 ± 3 62 ± 11b 4 ± 4 11 ± 7 373 ± 20 
SUMO1101 −52 ± 3 73 ± 6 8 ± 2 28 ± 3a 12 ± 3 35 ± 8b 28 ± 3a 12 ± 3 100 ± 15b 4 ± 8 13 ± 4 378 ± 17 
SUMO1101 75 −49 ± 4a 84 ± 5a 13 ± 3a 39 ± 3b 12 ± 3 22 ± 8b 37 ± 4b 10 ± 2 61 ± 8b 4 ± 3 11 ± 4 375 ± 19 
SUMO1101 250 −47 ± 6a 86 ± 5a 14 ± 4a 37 ± 4b 11 ± 4 21 ± 9b 35 ± 6b 9 ± 4 63 ± 9b 3 ± 4 12 ± 6 370 ± 24 
SUMO195 75 −60 ± 5 63 ± 6 6 ± 4 20 ± 3 13 ± 3 73 ± 7 20 ± 3 13 ± 3 249 ± 11 5 ± 4 13 ± 3 372 ± 15 
denatured SUMO1101 75 −57 ± 5 64 ± 3 5 ± 2 19 ± 4 12 ± 3 70 ± 8 20 ± 5 11 ± 4 240 ± 20 4 ± 4 13 ± 3 378 ± 18 
SENP1 250 −65 ± 5a 51 ± 5a 1 ± 2a 3 ± 4b 13 ± 4 100 ± 18b 1 ± 4b 10 ± 2 370 ± 23b 4 ± 4 13 ± 4 366 ± 21 
SENP1-C603S 250 −57 ± 5 64 ± 6 6 ± 3 20 ± 4 13 ± 4 72 ± 14 19 ± 6 9 ± 3 249 ± 17 4 ± 3 13 ± 3 388 ± 21 
Δ SENP-SUMO  16 ± 6 33 ± 8 12 ± 4 36 ± 5 0 ± 5 78 ± 20 36 ± 6 0 ± 3 309 ± 24 0 ± 5 0 ± 6 9 ± 28 

Neurons (Figs. 1 and 2) and cloned channels in CHO cells (Fig. 4) were studied in whole cell mode using filter and sampling frequencies of 5 and 25 kHz for voltage clamp studies and 1 and 10 kHz for current clamp recording. Voltage-dependent currents were studied after a P/5 leak subtraction. Stimulation protocols are described in the figure legends. Membrane potential and potassium currents were studied in solution A (in mM): 1.3 CaCl2, 0.5 MgCl2, 0.4 MgSO4, 3.56 KCl, 0.44 KH2PO4, 139.7 NaCl, 0.34 Na2HPO4, 5.5 glucose, and 10 HEPES, pH 7.4 with NaOH. Electrodes were filled with solution B (in mM): 136 KCl, 1 MgCl2, 2 K2ATP, 5 EGTA, and 10 HEPES, pH 7.2 with KOH. Sodium currents were studied with solution C in the bath (in mM): 130 NaCl, 5 CsCl, 2 CaCl2, 1.2 MgCl2, 5 glucose, and 10 HEPES, pH 7.4 with NaOH. The electrode solution D contained (in mM): 60 CsCl, 80 CsF, 10 EGTA, 1 CaCl2, 1 MgCl2, 5 Na2ATP, and 10 HEPES, pH 7.4 with CsOH. To determine V1/2, voltage evoking half-maximal conductance, and Vs, the slope of the curve, the normalized current was plotted against voltage and fitted to the Boltzmann equation, G = Gmax /(1+exp [−(V−V1/2)/Vs]), where Gmax is maximum conductance. IPEAK was determined at +50 mV. 8–20 cells were studied in each condition. ΔSENP-SUMO is the mean difference between exposure to SENP1 and 75 pM SUMO1101. Vm, resting membrane potential; Rin, input resistance; Ff, firing frequency.

a

Statistical difference determined by comparison to untreated cells using unpaired t tests, P < 0.01.

b

Statistical difference determined by comparison to untreated cells using unpaired t tests, P < 0.001.

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