Small-conductance chloride channels induced by cAMP,Ca2+, and hypotonicity in HT29 cells: ion selectivity,additivity and stilbene sensitivity

Previous studies in HT₂₉ cells utilizing the cellattached nystatin (CAN) method [Greger R, Kunzelmann K (1991) Pflügers Arch 419:209–211] have revealed that the Cl^– channels induced by cAMP or by increasing cytosolic Ca²⁺, e.g. by addition of ATP, and by hypotonic cell swelling share in common their conductance, which was so small in our studies [Kunzelmann et al. (1992) Pflügers Arch (in press)] that we could not resolve it at the single-channel level. This prompted the question whether these Cl^– conductances can be distinguished in terms of their ion selectivity and sensitivity towards inhibitors. Whether these pathways are additive or not was also examined. The present study utilized the whole-cell patch-clamp and the CAN methods. A total of 160 patches were studied. In whole-cell patches 8-(4-chlorophenylthio)-cAMP (cAMP, 0.1±1 mmol/l) induced a significant depolarization by 5 mV and a twofold increase in conductance (G) from 6.2±1.5 nS to 11.7±3.2 nS (n=15). Total replacement of Cl^– by Br^– and I^– in cAMP-treated cells hyperpolarized the membrane voltage (V) significantly from –35±2.8 to –39±3.4 and –45± 3.3 mV respectively, but had no detectable effect on G, which was 11.9±3.3 nS in the case of Br^– and 11.8± 3.3 nS in the case of I^–. Hence, the permselectivity of the cAMP pathway was I^–>Br^–>Cl^–, but the conductances for these anions were all indistinguishable. For ATP at 10–100 mol/l the depolarization was least with I^–: from –41±1.1 to –36±2.4mV, intermediate for Br^– to –25±1.6 mV, and largest for Cl^– to –20±1.8 mV (n=18). ATP increased G from 3.4±0.3 nS to 12.9±2.8 nS (Cl^–), to 12.9±2.8 nS (Br^–) and to 12.9±2.7 (I^–) (n=18). These data indicate that the ATP-induced anion channel has a permeability sequence of I^–>Br^–>Cl^–. The conductance for all three anions was identical. Hypotonic cell swelling by 160 mosmol/l induced a depolarization that was smallest for I^–, from –42±4 to –32±2.1 mV, intermediate for Br^–: –29±1.8mV, and similar for Cl^–: –28±2 mV (n=20). G was increased from 2.8±0.8 nS to 15±2.5nS in the case of Cl^–, to 15±2.5 nS for Br^– and to 16±2.6 nS for I^– (n=20). Therefore, all three pathways are indistinguishable with respect to their anion selectivity. All three pathways are insensitive towards low concentrations of 4-nitro-2-(3-phenylpropylamino)benzoate, but are all blocked by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid, with a half-maximal inhibition around 0.6 mmol/l. Finally, the possible additivity was examined in three permutations. ATP (0.1 mmol/l) alone (n=14) had a slightly but not significantly larger effect on conductance than the combination of ATP and cAMP (1 mmol/l, n=14) and the combination of ATP and hypotonicity (193 mosmol/l, n=13). Similarly, the effects of hypotonicity and cAMP (n=11) were not additive. These data indicate that all three pathways share common properties. Hence, it is suggested that all three pathways converge on the same small Cl^– channel.Supported by DFG Gr 480/10 and BMFT 01 GA 8816