HIGHLY COOPERATIVE CA2-3 MICROPERFUSION THROUGH A PATCH-CLAMP PIPETTE( ELEVATIONS IN RESPONSE TO INS(1,4,5)P)

To study the initial kinetics of Ins(1, 4, 5)P-3-induced [Ca2+](i) ele vations with a high time resolution and to avoid the problem of cell-t o-cell heterogeneity, we have used the combined patch-clamp/microfluor imetry technique. The mathematical description of the microperfusion o f Ins(1, 4, 5)P-3 and the subsequent Ca2+ release consists of a monoex ponential decay (cytosolic Ins(1,4, 5)P-3 concentration) and a Hill eq uation (Ins(1, 4, 5)P-3 dose-response curve). Two additional Hill equa tions and an integration were necessary to include a putative dependen ce of Ins(1, 4, 5)P-3-induced Ca2+ release on [Ca2+](i). Best-fitting analysis assuming [Ca2+](i)-independent Ca2+ release yielded Hill coef ficients between 4 and 12. The high cooperativity was also observed wi th the poorly metabolizable analog Ins(2, 4, 5)P-3 and was independent of extracellular [Ca2+]. Best-fitting analysis including a positive [ Ca2+](i) feedback suggested a cooperativity on the level of Ins(1, 4, 5)P-3-induced channel opening (n = 2) and an enhancement of Ins(1, 4, 5)P-3-induced Ca2+ release by [Ca2+](i). In summary, the onset kinetic s of Ins(1, 4, 5)P-3-induced [Ca2+](i) elevations in single HL-60 gran ulocytes showed a very high cooperativity, presumably because of a coo perativity on the level of channel opening and a positive Ca2+ feedbac k, but not because of Ca2+ influx or Ins(1, 4, 5)P-3 metabolism. This high cooperativity, acting in concert with negative feedback mechanism s, might play an important role in the fine-tuning of the cellular Ca2 + signal.