Inositol 1,3,4,5-tetrakisphosphate is essential for sustained activation of the Ca2+-dependent K+ current in single internally perfused mouse lacrimal acinar cells

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Abstract
Summary We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca2+-activated K+ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P3) (10–100 μm) evoked a transient increase in the Ca2+-sensitive K+ current that was independent of the presence of Ca2+ in the external solution. The transient nature of the Ins 1,4,5 P3 effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5mm 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P3, as well as with the stable analoguedl-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)3) (100 μm). Ins 1,3,4 P3 (50 μm) had no effect, whereas 50 μm Ins 2,4,5 P3 evoked responses similar to those obtained by 10 μm Ins 1,4,5 P3. A sustained increase in Ca2+-dependent K+ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P4) (10 μm) was added to the Ins 1,4,5 P3 (10 μm)-containing solution and this effect could be terminated by removal of external Ca2+. The effect of Ins 1,3,4,5 P4 was specifically dependent on the presence of Ins 1,4,5 P3 as it was not found when 10 μm concentrations of Ins 1,3,4 P3 or Ins 2,4,5 P3 were used. Ins 2,4,5 P3 (but not Ins 1,3,4 P3) at the higher concentration of 50 μm did, however, support the Ins 1,3,4,5 P4-evoked sustained current activation. Ins 1,3,4 P3 could not evoke sustained responses in combination with Ins 1,4,5 P3 excluding the possibility that the action of Ins 1,3,4,5 P4 could be mediated by its breakdown product Ins 1,3,4 P3. Ins 1,3,4,5 P4 also evoked a sustained response when added to an Ins 1,4,5 P(S)3-containing solution. Ins 1,3,4,5,6 P5 (50 μm) did not evoke any effect when administered on top of Ins 1,4,5 P3. In the absence of external Ca2+, addition of Ins 1,3,4,5 P4 to an Ins 1,4,5 P3-containing internal solution evoked a second transient K+ current activation. Readmitting external Ca2+ in the continued presence internally of Ins 1,4,5 P3 and Ins 1,3,4,5 P4 made the response reappear. We conclude that both Ins 1,4,5 P3 and Ins 1,3,4,5 P4 play crucial and specific roles in controlling intracellular Ca2+ homeostasis.

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