Highly cooperative Ca2+ elevations in response to Ins(1,4,5)P3 microperfusion through a patch-clamp pipette.
To study the initial kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations with a high time resolution and to avoid the problem of cell-to-cell heterogeneity, we have used the combined patch-clamp/microfluorimetry technique. The mathematical description of the microperfusion of Ins(1,4,5)P3 and the subsequent Ca2+ release consists of a monoexponential decay (cytosolic Ins(1,4,5)P3 concentration) and a Hill equation (Ins(1,4,5)P3 dose-response curve). Two additional Hill equations and an integration were necessary to include a putative dependence of Ins(1,4,5)P3-induced Ca2+ release on [Ca2+]i. Best-fitting analysis assuming [Ca2+]i-independent Ca2+ release yielded Hill coefficients between 4 and 12. The high cooperativity was also observed with the poorly metabolizable analog Ins(2,4,5)P3 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)P3-induced channel opening (n = 2) and an enhancement of Ins(1,4,5)P3-induced Ca2+ release by [Ca2+]i. In summary, the onset kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations in single HL-60 granulocytes showed a very high cooperativity, presumably because of a cooperativity on the level of channel opening and a positive Ca2+ feedback, but not because of Ca2+ influx or Ins(1,4,5)P3 metabolism. This high cooperativity, acting in concert with negative feedback mechanisms, might play an important role in the fine-tuning of the cellular Ca2+ signal.
Schrenzel
J
J
Infectious Diseases Division, University Hospital, Geneva, Switzerland. schrenze@dminovl.hcuge.ch
Demaurex
N
N
Foti
M
M
Van Delden
C
C
Jacquet
J
J
Mayr
G
G
Lew
D P
DP
Krause
K H
KH
D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D005456:Fluorescent Dyes; D016257:Fura-2; D018922:HL-60 Cells; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008433:Mathematics; D008832:Microchemistry; D008962:Models, Theoretical; D018408:Patch-Clamp Techniques; D010477:Perfusion / Q000379:methods; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
D002132:Calcium Radioisotopes; D005456:Fluorescent Dyes; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium; D016257:Fura-2