(Dys)regulation of epithelial chloride channels.

Chloride (Cl)-conducting channel proteins in plasma and intracellular membranes subserve a diversity of cellular functions, including neurotransmission, osmoregulation, pH regulation in organelles, acid secretion by parietal cells in the stomach, and salt absorption or secretion in exocrine glands (pancreas, sweat gland) and transport epithelia (trachea, intestine). With the notable exception of the y-aminobutyric acid (GABA)and glycine-gated neuronal C1channels [l], none of the anion-selective channel proteins have so far been purified, cloned and sequenced, hampering a structural comparison and classification of the various types of epithelial and non-epithelial Clchannels. However, the partial purification of epithelial Cl--channel proteins has been achieved recently by affinity chromatography of trachea and kidney membrane extracts on an immobilized indanyloxyacetic acid derivative belonging to a new class of potent and selective Cl--channel inhibitors [2]. In contrast, rapid progress has been made in the characterization and classification of anion-selective channels on the basis of biophysical criteria, following the introduction of patch-clamp and planar lipid bilayer techniques [3-5 1. The major anion conductance occurring in the apical membrane of all salt-secreting epithelial cell types analysed so far is a 30-50 pS outwardly rectifying channel characterized by the selectivity sequence C10,>NO,> I > Br> Cl> F> acetate& gluconate [3, 61. In intact cells, the gating of this apical Clchannel is triggered by a variety of intracellular signals including cyclic AMP, cyclic GMP (acting exclusively in intestinal epithelial cells), and Ca2+ [7, 81. In addition, the same C1channels can be activated non-physiologically and irreversibly by other manoeuvres, such a patch excision at depolarizing voltages and cell perfusion during whole-cell patch-clamp recording, possibly resulting in the removal of an endogenous Cl--channel inhibitor [3, 4, 91. The selective loss of Ca2+-, cyclic GMP-, but not of cyclic AMP-, responsive CI currents observed during maturation of Caco-2 colon carcinoma cells in culture [ 101 and the recent isolation of a T-84 colon carcinoma subclone displaying Ca2+-activable, but cyclic nucleotide-insensitive, C1currents (H. R. De Jonge, unpublished work), support a regulatory model in which each signal is capable of activating the C1channel independently of the other messengers through a common or different regulatory component. Hypothetically, cyclic nucleotide and Ca2+ regulation of ion channels may occur through: (i) a direct interaction with an allosteric modifier site on the channel protein; examples of this mechanism include the cyclic GMP-gated cation channel in rod and cone photoreceptors [ 111 and cyclic GMP inhibition of an amiloride-sensitive Na+ channel in renal inner medullary collecting duct cells [ 121; (ii) phosphorylation of the channel protein or regulatory subunit by cyclic AMP-, cyclic GMPor CaZ + -dependent protein kinases; (iii) cyclic nucleotideor Ca2 +-provoked fusion of channelcontaining vesicles with the apical membrane (‘channel recruitment’). Direct evidence in favour of the second mechanism has been obtained recently in patch-clamp studies of excised membrane patches from human tracheal cells [U, 131, lymphocytes [14], and colon carcinoma cells