Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin

Abstract

High-conductance apical K+ (BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K(+) losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K+ enrichment. We found that PKA activation using 50 micromol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 micromol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 micromol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 micromol/l Na+ orthovanadate and 10 micromol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K+ losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.

Fig. 1.

Effect of 50 μmol/l forskolin (Forsk) and 1 mmol/l IBMX on high-conductance apical K⁺ (BK) channel activity. A: responses in 7 cell-attached patches in surface colonocytes after 10-min exposure [140 mmol/l NaCl in bath and 145 mmol/l KCl in pipette, command voltage (V_com) = 0 mV]. B: single experiment from A showing a quiescent patch under basal conditions (command voltage = 0 mV), with stimulated channel activity at different command voltages. In this figure and Figs. 3, 5, and 6, dashed lines indicate closed channel current and downward deflections denote inward K⁺ current flow from pipette to cell. C: current-voltage relationship from experiment shown in B, indicating a single-channel conductance (g) of 135 pS.

Fig. 2.

Effect of PKA catalytic subunit on BK channel activity. A: responses in 10 excised inside-out patches in surface colonocytes after 15-min exposure to PKA catalytic subunit (200 U/ml) and 100 μmol/l ATP (140 mmol/l NaCl in bath and 145 mmol/l in pipette, command voltage = 0 mV). B: summary of the data from 10 excised inside-out patches showing time course of stimulation of channel activity by PKA catalytic subunit. P_o, single-channel open probability.

Fig. 3.

Effect of 2 μmol/l somatostatin (SOM) on BK channel activity. A: time course of SOM-induced inhibition of channel activity in 7 cell-attached patches in surface colonocytes (140 mmol/l NaCl in bath and 145 mmol/l in pipette, command voltage = 0 mV). Mean data ± SE are shown. *P < 0.02 and **P < 0.001 compared with t = 0 by ANOVA. B: representative 3-s segments from 30-s recordings from single patch containing 1 spontaneously active BK channel in the basal state (left) and after 25-min exposure to SOM. C: sustained inhibition of channel activity (P_o) produced by SOM during the entire 30-s recordings represented in B.

Fig. 4.

Effect of pertussis toxin (PTX) on SOM-induced inhibition of BK channel activity. Summary of channel activity before (t = 0) and at 5-min intervals after addition of 2 μmol/l SOM with (▪) and without (□) PTX pretreatment (200 ng/ml) for 18–24 h (n = 5 both groups, 140 mmol/l NaCl in bath and 145 mmol/l in pipette, command voltage = 0 mV). Mean data ± SE are shown. *P < 0.02 and **P < 0.001 compared with t = 0 by ANOVA.

Fig. 5.

Effect of 10 μmol/l okadaic acid (OA) on SOM-induced inhibition of BK channel activity. A: results from 6 cell-attached patches in surface colonocytes indicating that okadaic acid had no effect on SOM-induced inhibition of channel activity (140 mmol/l NaCl in bath and 145 mmol/l in pipette, command voltage = 0 mV). Mean data ± SE are shown. *P < 0.05 compared with t = 0 by ANOVA. B: representative recordings from a patch containing 1 spontaneously active BK channel in the basal state (top trace, P_o = 0.87), after 15-min pretreatment with okadaic acid (middle trace, P_o = 0.94), and 30 min after subsequent addition of SOM (bottom trace, P_o = 0.10).

Fig. 6.

Effect of 100 μmol/l Na⁺ orthovanadate (Orth) and 10 μmol/l potassium bisperoxo(1,10-phenanthroline) oxovanadate(V) (BpV) on SOM-induced inhibition of BK channel activity. A: results from cell-attached patches in surface colonocytes (140 mmol/l NaCl in bath and 145 mmol/l in pipette, command voltage = 0 mV) indicating that Na⁺ orthovanadate (▪; n = 6) and BpV (ο; n = 6) completely prevented the inhibition of channel activity produced by SOM alone (□; n = 4). Mean data ± SE are shown. *P < 0.05 and **P < 0.002 compared with t = 0 by ANOVA. B: representative recordings from a patch containing 4 spontaneously active BK channels in the basal state (top trace, P_o = 0.69), after 15-min pretreatment with Na⁺ orthovanadate (middle trace, P_o = 0.70), and 30 min after subsequent addition of SOM (bottom trace, P_o = 0.92). C: representative recordings from a patch containing 3 spontaneously active BK channels in the basal state (top trace, P_o = 0.93), after 15-min pretreatment with BpV (middle trace, P_o = 0.96), and 30 min after subsequent addition of SOM (bottom trace, P_o = 0.95).