Taurodeoxycholate activates potassium and chloride conductances via an IP₃-mediated release of calcium from intracellular stores in a colonic cell line (T84)

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca²⁺ concentration, (Ca²⁺)_i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl^- secretion in the 184 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to E_K and E_Cl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K⁺ and Cl^- currents with peak values of 385±41 pA and 98±28 pA, respectively (n = 12). The K⁺ and Cl^- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl^- secretion, induced no current response. The TDC-induced currents could be activated in Ca²⁺-free bathing solutions. Preincubation of cells with the Ca²⁺ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetra(acetoxymethy)-ester (20 μM), (BAPTA-AM), eliminated the K⁺ and Cl^- current responses, although the nonselective cation channel events were still present. Replacement of bath Na⁺ with NMDG⁺ inhibited the TDC-induced nonselective cation current but did not affect the K⁺ or Cl^- currents. TDC induced a transient (Ca²⁺)_i rise of 575±70 nM from a baseline of 71±5 nM(n = 15); thereafter, (Ca²⁺)_i either plateaued or oscillated. TDC-induced (Ca²⁺)_i oscillations were observed in the absence of bath Ca²⁺; however, removal of bath Ca²⁺ during the TDC response caused (Ca²⁺)_i to return to near baseline values. Simultaneous K⁺ current and (Ca²⁺)_i measurements confirmed that the initial nonselective cation current was independent of (Ca²⁺)_i, while K⁺ current oscillations were in phase with the (Ca²⁺)_i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP₃) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca²⁺ -activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca²⁺-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl^- secretion by activating K⁺ and Cl^- conductances via an IP₃-mediated release of Ca²⁺ from intracellular stores.