Regulation of Ca2+-dependent Cl− conductance in a human colonic epithelial cell line (T84): cross-talk between Ins(3456)P4 and protein phosphatases

摘要

class="enumerated" style="list-style-type:decimal">We have studied the regulation of whole-cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4). 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)P4 (i.e. F2-Ins(3,4,5,6)P4).Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current (ICl,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 ICl,CaMK remained near its maximum value for ≥ 14.3 ± 0.6 min.F2-Ins(3,4,5,6)P4 inhibited ICl,CaMK (IC50 = 100 μm) stereo-specifically, since its enantiomer, F2-Ins(1,4,5,6)P4 had no effect at < = 500 μm. Dose-response data (Hill coefficient = 1.3) showed that F2-Ins(3,4,5,6)P4 imitated only the non-co-operative phase of inhibition by Ins(3,4,5,6)P4, and not the co-operative phase.Ins(3,4,5,6)P₄ was prevented from blocking I_Cl,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)P₄ to function. The IC₅₀ values indicate that more than one species of phosphatase was required. One of these may be PP1, since F₂-Ins(3,4,5,6)P₄-dependent current blocking was inhibited by okadaic acid and microcystin with IC₅₀ values of 70 nm and 0.15 nm, respectively.