Small-conductance chloride channels induced by cAMP, Ca2+, and hypotonicity in HT29cells: ion selectivity, additivity and stilbene sensitivity
✍ Scribed by R. Kubitz; R. Warth; N. Allert; K. Kunzelmann; R. Greger
- Publisher
- Springer
- Year
- 1992
- Tongue
- English
- Weight
- 889 KB
- Volume
- 421
- Category
- Article
- ISSN
- 0031-6768
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✦ Synopsis
Previous studies in HT29 cells utilizing the cellattached nystatin (CAN) method [Greger R, Kunzelmann K (1991) Pftfigers Arch 419:209-211] have revealed that the C1-channels induced by cAMP or by increasing cytosolic Ca 2 +, 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) Pflfigers Arch (in press)] that we could not resolve it at the single-channel level. This prompted the question whether these C1-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-(4chlorophenylthio)-cAMP (cAMP, 0.1 _+ 1 mmol/1) induced a significant depolarization by 5 mV and a twofold increase in conductance (G) from 6.2+1.5nS to 11.7 _+ 3.2 nS (n = 15). Total replacement of C1-by Brand 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-> CI-, but the conductances for these anions were all indistinguishable. For ATP at 10-100 gmol/1 the depolarization was least with I-" from -41 i 1.1 to -36_+2.4mV, intermediate for Br-to -25_+ 1.6 mV, and largest for C1-to -20 + 1.8 mV (n = 18). ATP increased G from 3.4 • 0.3 nS to 12.9 • 2.8 nS (C1-), 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-> C1-. The conductance for all three anions was identical. Hypotonic cell swelling by 160 mosmol/1 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 * Supported by DFG Gr 480/J0 and BMFT 01 GA 8816 Correspondence to. R. Greger
C1-" -28_2mV
(n = 20). G was increased from 2.8_+0.8nS to 15_+2.5nS in the case of CI-, to I5 _ 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-(3phenylpropylamino)benzoate, but are all blocked by 4,4'diisothiocyanatostilbene-2,2'-disulphonic acid, with a half-maximal inhibition around 0.6 mmol/1. Finally, the possible additivity was examined in three permutations. ATP (0.1 retool/l) alone (n = 14) had a slightly but not significantly larger effect on conductance than the combination of ATP and cAMP (l retool/l, n = 14) and the combination of ATP and hypotonicity (193 mosmol/1, n = 13). Similarly, the effects ofhypotonicity 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 C1-channel.