Inositol 3,4,5,6-Tetrakisphosphate Inhibits the Calmodulin-dependent Protein Kinase II-activated Chloride Conductance in T84 Colonic Epithelial Cells

Abstract

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.

Anoctamin 1 (Tmem16A) Ca 2 + -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

“…Annexin A1 is intriguing because various annexins have been shown to regulate native Ca 2+ -activated Cl − channels. For example, annexin IV inhibits calmodulin-sensitive Cl − channels (37,38) as well as Ca 2+ -activated Cl − channels (39). In endothelial cells the annexin II-S100A10 complex might be involved in regulation of the swelling-activated Cl − channel (40) and cystic fibrosis transmembrane conductance regulator (41).…”

The Structural and Functional Versatility of Inositol Phosphates

“…This effect was mediated downstream of CaMK II, the activity of which was not affected by Ins(3,4,5,6)P 4 . This is a very specific effect, in T84 cells and elsewhere: Ins(l,3,4)P 3 , Ins(l,3,4,6)P 4 , Ins(l,3,4,5)P 4 , Ins(l,4,5,6)P 4 and Ins(l,3,4,5,6)P 5 all have no effect upon CI" current even when added at concentrations 15 times greater than the IC^ value for Ins(3,4,5,6)P 4 (110)(111)(112).…”

“…Autophosphorylated and constitutively active CaMK Π was perfused into the cell through the patch pipette. This brought about a 20-fold increase in the CI" current (110). The CaMK Π-activated current was completely blocked by 10 μΜ Ins(3,4,5,6)P 4 , even though the polyphosphate had no effect upon CaM KII activity itself.…”

“…First, Ismailov et al, (Ill) found that nanomolar concentrations of Ins(3,4,5,6)P 4 blocked Cl Ca . In contrast, 2-10 μΜ Ins(3,4,5,6)P 4 is required inside cells to inhibit CI" current (65,110,112). Second, in the bilayer experiments a single site dose/response relationship for Ins(3,4,5,6)P 4 was obtained, without any evidence for co-operativity (777).…”

“…Second, in the bilayer experiments a single site dose/response relationship for Ins(3,4,5,6)P 4 was obtained, without any evidence for co-operativity (777). Ins(3,4,5,6)P 4 behaves differentiy inside cells, where a particularly steep slope in the dose/response curve suggests a highly co-operative mechanism of action (110,112). These quantitative differences in the results obtained in the two experimental paradigms suggest that important auxiliary factors also participate in the action of Ins(3,4,5,6)P 4 in vivo.…”