Smooth muscle aldolase C-bound inositol 1,4,5-trisphosphate studied in vitro under physiological conditions

Abstract

Our goal was to quantitate inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) binding to aldolase C tetramer (aldolase 4) and its displacement by inositol 1,3,4-trisphosphate (Ins(1,3,4)P 3) under conditions which approximated the in vivo state. Anions were found to have major effects. Decreasing [KCl] from 100 to 10 mM, at 0°C and pH 7.0, increased maximal Ins(1,4,5)P 3 binding to 1.0 to 2.4 mol per mol aldolase 4. At 10 and 30 mEq/l [Cl −], an additional high affinity site was detected ( K ds=0.43 and 0.86 μM, respectively). Increasing concentrations of other anions (SO 2− 4, propanoate −, HCO − 3, acetate −) also inhibited binding, but effects would be minimal at concentrations of these anions present in the cytoplasm of living cells. Ins(1,3,4)P 3 displacement of aldolase C-bound Ins(1,4,5)P 3 was sensitive to [Cl −]; at 30 mEq/l [Cl −] and 37°C, Ins(1,3,4)P 3 released 20% of bound Ins(1,4,5)P 3 at concentrations of 100 nM. Changing temperature from 0° to 37°C increased K ds for Ins(1,4,5)P 3binding. Changes in free [Ca 2+], [Mg 2+], [Na +] and [K +] and changes in osmolality had no effect on Ins(1,4,5)P 3 binding to aldolase C. In vivo Ins(1,4,5)P 3–aldolase 4 binding at 30 mEq/l [Cl −] and 37°C were calculated for different [Ins(1,4,5)P 3] free over the range 0.2 to 1.0 μM. For different cytoplasmic [Ins(1,4,5)P 3] free, Ins(1,4,5)P 3 binding to aldolase 4 was sufficient, if acutely released, to nearly double cytoplasmic [Ins(1,4,5)P 3] free. We proposed a schema whereby release of aldolase C-bound Ins(1,4,5)P 3 evoked by Ins(1,3,4)P 3 amplifies effects of phospholipase C-formed Ins(1,4,5)P 3.