REGULATION OF CA2-DEPENDENT CL- CONDUCTANCE IN A HUMAN COLONIC EPITHELIAL-CELL LINE (T-84) - CROSS-TALK BETWEEN INS(3,4,5,6)P-4 AND PROTEINPHOSPHATASES()

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-bisfl uoro-Ins(3,4,5,6)P-4 (i.e. F-2-Ins(3,4,5,6)P-4). 2. Calmodulin-depende nt protein kinase II (CaMKII) increased chloride current 20-fold. This current (I-Cl,I-CaMK) continued for 7 +/- 1.2 min before its deactiva tion, or running down, by approximately 60%. This run-down was prevent ed by okadaic acid, whereupon I-Cl,I-CaMK remained near its maximum va lue 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 equa l 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 inhib ition 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 f or 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, sin ce F-2- Ins(3,4,5,6)P-4-dependent current blocking was inhibited by ok adaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, resp ectively.