Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). I. Single-channel properties.
The patch-clamp technique was combined with camera-based intracellular Ca2+ concentration ([Ca2+]i) imaging to identify the single-channel basis of the Ca(2+)-dependent Cl- conductance in human colonic adenocarcinoma cells (HT-29). Cl- channels were activated when membrane patches were excised into solutions containing high (1 microM) Ca2+ concentrations. Their single-channel conductance, measured by amplitude histogram analysis, averaged 13 pS at -90 mV and 16 pS at +90 mV membrane potential (MP). In multiple channel patches, Cl- currents showed properties similar to Ca(2+)-activated whole cell currents: outward rectification and time-dependent activation at depolarizing MP. Channel activity disappeared shortly after patch excision from the cell. In cell-attached patches, Cl- channel opening was infrequent at resting [Ca2+]i values (96 +/- 18 nM), but when [Ca2+]i was increased by the Ca2+ ionophore ionomycin (1 microM), Cl- channels were activated with a time course that paralleled the [Ca2+]i rise. Repetitive ionophore exposure produced equivalent rises in [Ca2+]i, but the corresponding Cl- channel activity became progressively reduced. The Ca(2+)-mediated agonist neurotensin (50 nM) elicited a transient Cl- channel activation that preceded the generalized cellular [Ca2+]i rise. Channel activation with neurotensin occurred in the absence of pipette Ca2+ but was abolished by preloading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus, in response to the Ca(2+)-mediated agonist neurotensin, Cl- channel activation results from Ca2+ mobilization from intracellular pools localized within the vicinity of the plasma membrane. The Ca2+ dependency, voltage sensitivity, and kinetics of this 15-pS Cl- channel indicate that it is the basis of the whole cell Ca(2+)-activated Cl- current.
Morris
A P
AP
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005.
Frizzell
R A
RA
D000230:Adenocarcinoma; D002118:Calcium / Q000502:physiology*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D008536:Meglumine / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D009496:Neurotensin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D009496:Neurotensin; D004533:Egtazic Acid; D015759:Ionomycin; D008536:Meglumine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium