SMALL-CONDUCTANCE CHLORIDE CHANNELS INDUCED BY CAMP, CA2+, AND HYPOTONICITY IN HT(29) CELLS - ION SELECTIVITY, ADDITIVITY AND STILBENE SENSITIVITY

Previous studies in HT29 cells utilizing the cell-attached nystatin (CAN) method [Greger R, Kunzelmann K (1991) Pflugers 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) Pflugers 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 mumol/l the depolarization was least with I-: from -41 +/- 1.1 to -36 +/- 2.4 mV, 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 +/- 1.8 MV, and similar for Cl-: -28 +/- 2 mV (n = 20). G was increased from 2.8 +/- 0.8 nS to 15 +/- 2.5 nS in the case of Cl-, to 15 +/- 2.5 nS for Br- and to 16 +/- 2.6 nS for 1- (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 three pathways share common properties. Hence, it s suggested that all three pathways converge on the same small Cl- channel.