Modulation of the hyperpolarization-activated Cl- current in human intestinal T84 epithelial cells by phosphorylation.
1. Hyperpolarization-activated Cl- currents (ICl,hyp) were investigated in the T84 human adenocarcinoma cell line, using the patch-clamp whole-cell configuration. 2. During whole-cell recording with high-chloride and ATP-containing internal solutions, hyperpolarizing jumps from a holding potential of 0 mV elicited slow inward current relaxations, carried by Cl- and detected at membrane potentials more negative than -40 mV. Analysis of the relative permeabilities to monovalent anions gave the following sequence: Cl- > Br- > I- > glutamate. 3. ICl,hyp was partially inhibited by 1 mM diphenylamine-2-carboxylic acid or 0.1 mM 5-nitro-2-(3-phenylpropylamino)-benzoate, and was completely blocked by Cd2+ (> 300 microM). It was insensitive to 1 mM external 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid or 1 mM Ba2+. 4. ICl,hyp was inhibited by external application of 500 microM cptcAMP (8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate) or 500 nM of the protein kinase C activator, phorbol 12-myristate, 13-acetate. 5. (i) Omission of ATP from the pipette solution, (ii) ATP replacement by the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate, and (iii) inhibition of protein kinase C by staurosporine or calphostin C accelerated the activation kinetics of the current and increased its amplitude, but did not alter its pharmacological properties. 6. We conclude that hyperpolarization-activated Cl- channels similar to those of ClC-2 channels (mammalian homologue of Torpedo chloride channel ClC-0) are present in T84 cells, and that their gating properties are modulated by phosphorylation.
Fritsch
J
J
CNRS URA 583, Hôpital des Enfants Malades, Paris, France.
Edelman
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology*; D004848:Epithelium / Q000502:physiology; D006801:Humans; D007422:Intestines / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation
D018118:Chloride Channels; D000255:Adenosine Triphosphate