G proteins activate ionic conductances at multiple sites in T84 cells

We examined the role of G proteins in activation of ionic conductances in isolated T84 cells during cholinergic stimulation. When cells were whole cell voltage clamped to the K⁺ equilibrium potential (E(K)) or Cl^- equilibrium potential (E(Cl)) under standard conditions, the cholinergic agonist, carbachol, induced a large oscillating K⁺ current but only a small inward current. Addition of the GDP analogue, guanosine 5'-O-(2- thiodiphosphate), to pipettes blocked the ability of carbachol to activate the K⁺ current. Addition of the nonhydrolyzable GTP analogue, guanosine 5'- O-(3-thiotriphosphate) (GTPγS), to pipettes stimulated large oscillating K⁺ and inward currents. This occurred even when Ca²⁺ was absent from the bath but not when the Ca²⁺ chelator, ethylene glycol-bis(β-aminoethyl ether)- N,N,N',N'-tetraacetic acid, was added to pipettes. When all pipette and bath K⁺ was replaced with Na⁺ and cells were voltage clamped between E(Na) and E(Cl), GTPγS activated oscillating Na⁺ and Cl^- currents. Finally, addition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] to pipettes activated large oscillating K⁺ currents but only small inward currents. These results suggest that a carbachol-induced release of Ca²⁺ from intracellular stores is activated by a G protein through the phospholipase C-Ins(1,4,5)P₃ signaling pathway. In addition, this or another G protein activates Cl^- current by directly gating Cl^- channels to increase their sensitivity to Ca²⁺.