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Regulation of Ca 2+ ‐dependent Cl − conductance in a human colonic epithelial cell line (T 8 4 ): cross‐talk between Ins(3,4,5,6) P 4 and protein phosphatases
Author(s) -
Xie Weiwen,
Solomons Kevin R. H.,
Freeman Sally,
Kaetzel Marcia A.,
Bruzik Karol S.,
Nelson Deborah J.,
Shears Stephen B.
Publication year - 1998
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1998.661bj.x
Subject(s) - okadaic acid , phosphatase , chemistry , camk , threonine , calmodulin , biochemistry , serine , phosphorylation , protein kinase a , enzyme , autophosphorylation
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,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. 3 F 2 ‐Ins(3,4,5,6) P 4 inhibited I Cl,CaMK (IC 50 = 100 μM) stereo‐specifically, since its enantiomer, F 2 ‐Ins(1,4,5,6) P 4 had no effect at <= 500 μM. 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 4 was prevented from blocking I Cl,CaMK by okadaic acid (IC 50 = 1.5 nM) and microcystin (IC 50 = 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 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 nM and 0.15 nM, respectively.