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

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 ). Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current ( I Cl,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,CaMK remained near its maximum value for ≥ 14.3 ± 0.6 min. F 2 -Ins(3,4,5,6) P 4 inhibited I Cl,CaMK (IC 50 = 100 μ m ) stereo-specifically, since its enantiomer, F 2 -Ins(1,4,5,6) P 4 had no effect at < = 500 μ 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. Ins(3,4,5,6) P 4 was prevented from blocking I Cl,CaMK by okadaic acid (IC 50 = 1.5 n m ) and microcystin (IC 50 = 0.15 n m ); these data lead to the novel conclusion that, in situ , protein phosphatase activity is essential for Ins(3,4,5,6) P 4 to function. The IC 50 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 IC 50 values of 70 n m and 0.15 n m , respectively.