Separate Cl- conductances activated by cAMP and Ca2+ in Cl(-)-secreting epithelial cells.

Abstract

We studied the cAMP-and Ca2+-activated secretory Cl1 conductances in the Cl--secreting colonic epithelial cell line T84 using the whole-cell patch-clamp technique. Cl-and K+ currents were measured under voltage damp. Forskolin or cAMP increased Cl-current 2-15 times with no change in K+ current. The current-voltage relation for cAMPactivated Cl-current was linear from -100 to +100 mV and showed no time-dependent changes in current during voltage pulses. Ca2l ionophores or increased pipette Ca2' increased both Cl-and K+ currents 2-30 times. The Ca2+-activated Clcurrent was outwardly rectified, activated during depolarizing voltage pulses, and inactivated during hyperpolarizing voltage pulses. Addition of ionophore after forskolin further increased Cl-conductance 1.5-5 times, and the current took on the time-dependent characteristics of that stimulated by Ca2 . Thus, cAMP and Ca2+ activate Cl-conductances with different properties, implying that these second messengers activate different Cl-channels or that they induce different conductive and kinetic states in the same Cl-channel.Secretion of salt and water across many epithelia is driven by a secondary active Cl-transport mechanism in which Clleaves the cell across the apical membranes by diffusion (1 by cAMP, and the other is activated by Ca2W. MATERIALS AND METHODSCell Culture. T84 cells were cultured as described (6). Cells were used 1-3 days after plating on collagen-coating coverslips. Individual cells and cells in islands were studied, but the cells in small groups were more frequently responsive to secretagogues than were isolated cells. Voltage-clamp artifacts arising from cell-to-cell coupling in islands of cells were insignificant, as evidenced by the small capacitive transients remaining after capacitance compensation.Whole-Cell Current Recordings. Standard whole-cell tightseal patch-clamp techniques were used (7, 8). The cells were maintained at 37°C by a constant-temperature microscope stage modified to hold the bath chamber and offer low electrical noise (model 5001, Micro Devices, Jenkintown, PA). At this temperature, the responsiveness of the cells to secretagogues, particularly to cAMP-dependent agonists, was improved.Increases in Cl-and K+ conductances are the major electrical events detected during stimulation of Cl-secretion (1). Accordingly, bath-pipette ion gradients were chosen so that transmembrane Cl-and K+ currents could be monitored independently at clamp voltages equal to the reversal potentials for these ions. The pipette solution was 115 mM KCI/25 mM N-methyl-D-glucamine (NMDG) glutamate/0.5 mM EGTA/0.19 mM CaCl2 (100 nM free Ca2+)/2 mM MgCl2/2 mM Na2ATP/0.05 mM Na3GTP/5 mM Hepes, adjusted to pH 7.2 with KOH. The bath solution was 115 mM NaCl/40 mM NMDG glutamate/5 mM potassium glutamate/2 mM MgCl2/1 mM CaC12/5 mM Hepes, adjusted to pH 7.2 with NaOH. Bath Na+ or Cl-concentrations were reduced by substituting NMDG chloride or sodium glutamate for NaCl. When Na+ and K+-free solutions were used, Na+ and K+ were replaced by NMDG...

A non‐selective cation current activated via the multifunctional Ca(2+)‐calmodulin‐dependent protein kinase in human epithelial cells.

“…1B b). The timedependent activation of outward currents during depolarizing voltage clamp steps is characteristic of the calcium-activated chloride current (IcI(ca)) previously observed in T84 cells (Cliff & Frizzell, 1990;Anderson & Welsh, 1991) and other epithelia (Morris, Kirk & Frizzell, 1990;Anderson & Welsh, 1991;Chan, Goldstein & Nelson, 1992). During steps to hyperpolarized membrane potentials, a modest time-dependent decay of the inward current was typically observed.…”

Antagonism of kinin effects on epithelia by Hoe 140: apparently competitive and non‐competitive interactions

“…For example, apical chloride channels can be Ca2"-sensitive (Cliff & Frizzell, 1990). Alternatively an increase in Ca2" opens basolateral Ca2"-sensitive K'channels, with consequent hyperpolarization and an increase in the electrical gradient for chloride efflux from the cell.…”

Volume‐activated Cl− secretion and transepithelial vinblastine secretion mediated by P‐glycoprotein are not correlated in cultured human T84 intestinal epithelial layers

“…and volume-activated CI-conductances have all been demonstrated, and this is correlated with the existence of separate Clchannels with distinct kinetic characteristics [2,[7][8][9]. In intact Ts4 epithelial layers, basal short circuit current (SCC) is minimal, but secretagogues such as vasoactive intestinal peptide (VIP) stimulate an inward transepithelial SCC.…”