Regulation of Ca2+-dependent Cl- conductance in a human colonic epithelial cell line (T84):: Cross-talk between Ins(3,4,5,6)P4 and protein phosphatases

1. We have studied the regulation of whole-cell chloride current in T-84 colonic epithelial cells by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P-4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1,2-bisdeoxy-1,2-bisfluoro-Ins(3,4,5,6)P-4 (i.e. F-2-Ins(3,4,5,6)P-4). 2. Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current (I-Cl,I-CaMK) continued for 7 +/- 1.2 min before its deactivation, or running down, by approximately 60%. This run-down was prevented by okadaic acid, whereupon I-Cl,I-CaMK remained near its maximum value for greater than or equal to 14.3 +/- 0.6 min. 3. F-2-Ins(3,4,5,6)P-4 inhibited I-Cl,I-CaMK (IC50 = 100 mu M) stereo-specifically since its enantiomer, F-2-Ins(1,4,5,6)P-4 had no effect at less than or equal to 500 mu M. Dose-response data (Hill coefficient = 1.3) showed that F-2-Ins(3,4,5,6)P-4 imitated only the non-co-operative phase of inhibition by Ins(3,4,5,6)P-4, and not the co-operative phase. 4. Ins(3,4,5,6)P, was prevented from blocking I-Cl,I-CaMK by okadaic acid (IC50 = 1.5 nM) and microcystin (IC50 = 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P, to function. The IC,, values indicate that more than one species of phosphatase was required. One of these may be PP1, since F-2- Ins(3,4,5,6)P-4-dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.