Volume 122, Issue 4, Pages 994-1006 (April 2002) SNAP-25, a SNARE protein, inhibits two types of K+ channels in esophageal smooth muscle  Junzhi Ji, Anne Marie F. Salapatek, Helena Lau, Guotang Wang, Herbert Y. Gaisano, Nicholas E. Diamant  Gastroenterology  Volume 122, Issue 4, Pages 994-1006 (April 2002) DOI: 10.1053/gast.2002.32412 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Localization of SNAP-25 and α-actin in feline esophageal circular smooth muscle cells by confocal immunofluorescence microscopy. (A and B) Esophageal circular muscle cells were double labeled with (A) mouse anti–α-actin and (B) rabbit anti–SNAP-25. Arrows indicate the abundance of SNAP-25 on the plasma membrane that colocalizes with α-actin. (C and D) Double labeling of (C) mouse anti–α-actin and (D) anti–SNAP-25, which was preincubated with the full-length SNAP-25 protein. (D) Preincubation with the full-length SNAP-25 protein completely blocked the signal of SNAP-25 (bar in D = 50 μm). Similar results were obtained in 3 sets of experiments. Gastroenterology 2002 122, 994-1006DOI: (10.1053/gast.2002.32412) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Inhibitory effect of SNAP-25 on outward currents of esophageal circular muscle cells. (A) A family of outward currents were induced by 250-ms voltage steps ranging from −50 mV to 60 mV. The holding potential was −50 mV. GST–SNAP-25 (10−8 mol/L) was microinjected into the cell via patch pipette. Current traces were obtained immediately after membrane rupture (left) and 10 minutes thereafter (middle). I-V relationships of steady-state currents before (○, n = 8) and 10 minutes after (●, n = 8) the diffusion of GST–SNAP-25 (right). Each point represents mean ± SEM. *P < 0.05, **P < 0.01 against control. GST–SNAP-25 reduced the outward current. (B) The same protocol as (A) was performed in another set of experiments except that 10−8 mol/L GST alone was included in the pipette solution instead of GST–SNAP-25. GST itself had no effect on the outward current (n = 4). (A and B) Dotted lines are the 0 current level. (C) Dose-dependent inhibition of outward currents by GST–SNAP-25 (10−11 to 10−7 mol/L in the pipette solution) assessed by depolarization to 30 mV from a holding potential of −50 mV. Steady-state outward current amplitudes (I) obtained 10 minutes after the diffusion of GST–SNAP-25 were normalized to those recorded just after breakthrough (Icontrol). There is a dose-dependent decrease in the outward current. Each point represents mean ± SEM. *P < 0.05, **P < 0.01 against control. The number of cells studied at each concentration was shown in parentheses. Gastroenterology 2002 122, 994-1006DOI: (10.1053/gast.2002.32412) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Isolation of KCa and KV from outward currents. (A) A family of outward currents were induced by 250-ms voltage steps from −50 mV to 60 mV from a cell before adding drugs (control, left), after adding 1 mmol/L TEA (middle), and after further adding 5 mmol/L 4-AP (right). The holding potential was −50 mV. TEA reduced the outward current by approximately 40% and 4-AP further reduced the majority of the remaining current. Dotted lines are the 0 current level. (B) I-V relationships were determined by plotting amplitudes of steady-state outward currents against step voltages. Each point represents mean ± SEM. Total outward currents were obtained before adding blockers (○, control, n = 6). Values for large-conductance Ca2+-activated K+ currents (KCa) (▴, n = 6) were calculated from total outward currents by subtracting 1 mmol/L TEA-insensitive currents; values for delayed rectifier K+ currents (KV) (●, n = 6) were the 1 mmol/L TEA-insensitive currents minus 4-AP–insensitive currents. Gastroenterology 2002 122, 994-1006DOI: (10.1053/gast.2002.32412) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 SNAP-25 inhibits the KCa current. (A) TEA-sensitive outward currents (left) from a control cell in the absence of (○) and the presence (●) of 1 mmol/L TEA. Decrease in outward current (middle) from a GST–SNAP-25 (10−8 mol/L)–injected cell, recordings taken 10 minutes after the diffusion of GST–SNAP-25 in the absence (○) and the presence (●) of 1 mmol/L TEA. Data were also obtained in control cells 10 minutes after membrane rupture. The decrease in KCa (1 mmol/L TEA-sensitive) current densities from 6 control cells (n = 6) and 6 GST–SNAP-25–injected cells (n = 6) (right). (B) Same experimental protocol as used in (A) except that 300 nmol/L IbTX was added to the bath solution instead of 1 mmol/L TEA. The IbTX-sensitive KCa current was similarly decreased when GST–SNAP-25 was injected into cells. Dotted lines are the 0 current level. Current amplitudes are normalized by cell capacitance to eliminate the influence of cell size variance. Each bar represents mean ± SEM. *P < 0.05 against control. Gastroenterology 2002 122, 994-1006DOI: (10.1053/gast.2002.32412) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 SNAP-25 inhibits the KV current. (A) Outward currents were obtained by 250-ms depolarization to 30 mV from isolated muscle cells immersed in the bath solution containing 1 mmol/L TEA and microinjected with 10−8 mol/L GST–SNAP-25. The holding potential was −50 mV. The decrease in outward current (left) from immediately after breakthrough (○, control) to 10 minutes thereafter (●). At the end of experiments, 5 mmol/L 4-AP was added to the bath solution to determine the amplitudes of 4-AP–insensitive currents (not shown). KV currents were determined from outward currents subtracting 4-AP–insensitive currents. The decrease in KV current density (right) from 6 cells injected with 10−8 mol/L GST–SNAP-25 immediately after rupture (control, n = 6) and 10 minutes thereafter (GST–SNAP-25, n = 6). (B) 4-AP–sensitive outward current (left) in a control cell in the absence of (○) and the presence (●) of 5 mmol/L 4-AP. We began recordings in control cells 10 minutes after breakthrough. The decrease in 4-AP–sensitive outward current (middle) in a GST–SNAP-25 (10−8 mol/L)–injected cell, recordings taken 10 minutes after the diffusion of GST–SNAP-25 in the absence (○) and the presence (●) of 5 mmol/L 4-AP. KV (4-AP sensitive) current densities (right) from control cells (n = 6) and GST–SNAP-25–injected cells (n = 6). (C) Same experimental protocol as used in (B) except that 300 nmol/L DTX was added to the bath solution instead of 5 mmol/L 4-AP. The DTX-sensitive KCa current was also decreased when GST–SNAP-25 was injected into cells (n = 6) but to a lesser degree than the 4-AP–sensitive current. Dotted lines are the 0 current level. Current amplitudes are normalized by cell capacitance to eliminate the influence of cell size variance. Each bar represents mean ± SEM. *P < 0.05, **P < 0.01 against control. Gastroenterology 2002 122, 994-1006DOI: (10.1053/gast.2002.32412) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Increase in outward currents after cleavage of endogenous SNAP-25 by BoNT/A. (A) A family of outward currents were induced by 250-ms voltage steps ranging from −50 mV to 60 mV. The holding potential was −50 mV. BoNT/A (100 nmol/L) was microinjected into the cell via patch pipette. Current traces were obtained immediately after membrane rupture (left) and 20 minutes thereafter (middle). I-V relationships of steady-state currents before (○, n = 6) and 20 minutes after (●, n = 6) the diffusion of BoNT/A (right). Each point represents mean ± SEM. *P < 0.05 against control. (B) Alteration of the outward current with time in 2 representative cells treated with BoNT/A (100 nmol/L, ●) or its inactive form (○), respectively. Outward currents were generated by depolarization to 30 mV from a holding potential of −50 mV. Steady-state outward current amplitudes (I) recorded at different times were normalized to that obtained just after the rupture of membrane (I0). Gastroenterology 2002 122, 994-1006DOI: (10.1053/gast.2002.32412) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Effects of SNAP-25 on voltage-dependent activation of K+ currents. Protocols were described in text as well as values for V½. Data presented as mean ± SEM were collected from cells injected with 10−8 mol/L GST–SNAP-25 just after breakthrough (○, control) and 10 minutes thereafter (●). GST–SNAP-25 shifted the activation curve to the right (A) in cells bathed in the standard external solution (n = 4) and (C) in cells bathed in 5 mmol/L 4-AP (n = 4), but (B) had no effect on that of cells bathed in 300 nmol/L IbTX (n = 3). Gastroenterology 2002 122, 994-1006DOI: (10.1053/gast.2002.32412) Copyright © 2002 American Gastroenterological Association Terms and Conditions