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 int racellular Ca2+ concentration, (Ca2+)i, were used to investigate the m echanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in th e T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to E(K) and E(Cl). Initi ally, TDC (0.75 mM) stimulated inward nonselective cation currents tha t were composed of discrete large conductance single-channel events. T his 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 returne d to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid th at does not stimulate colonic Cl- secretion, induced no current respon se. The TDC-induced currents could be activated in Ca2+-free bathing s olutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminop henoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)ester (2 0 muM), (BAPTA-AM), eliminated the K+ and Cl- current responses, altho ugh the nonselective cation channel events were still present. Replace ment of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cat ion current but did not affect the K+ or Cl- currents. TDC induced a t ransient (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+), oscillations were observed in the absence of bath Ca2+; howeve r, removal of bath Ca2+ during the TDC response caused (Ca2+)i to retu rn to near baseline values. Simultaneous K+ current and (Ca2+)i measur ements confirmed that the initial nonselective cation current was inde pendent of (Ca2+)i, while K+ current oscillations were in phase with t he (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accum ulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) d uring TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell re cordings, except the nonselective cation current was prolonged. When h eparin (1 mg/ml) was added to the pipette under these conditions, the Ca2+-activated currents were inhibited, but the nonselective cation cu rrents were unaffected. These data suggest that TDC induces a Ca2+-ind ependent nonselective cation conductance, perhaps by directly permeabi lizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intr acellular stores.