J. Biol. Chem., Vol. 269, Issue 26, 17561-17571, 07, 1994
Rapid filtration studies of the effect of cytosolic Ca2+ on inositol 1,4,5-trisphosphate-induced 45Ca2+ release from cerebellar microsomes
L Combettes, Z Hannaert-Merah, JF Coquil, C Rousseau, M Claret, S Swillens and P Champeil
Unite de Recherche U274, Institut National de la Sante et de la Recherche Medicale, Universite Paris-Sud, Orsay, France.
Using microsomal membrane vesicles derived from sheep cerebellum, we
measured the rate of inositol 1,4,5-trisphosphate (InsP3)-dependent 45Ca2+
efflux from 45Ca(2+)-loaded compartments during rapid perfusion with a
medium containing InsP3 and various concentrations of free 40Ca2+ on the
cytosolic side (pH 7.1, 5 mM Mg2+, in the absence of ATP at 20 degrees C).
At 0.15 microM InsP3 and pCa 6.5, half-45Ca2+ release was attained within
less than 200 ms. At low Ca2+ concentrations, the initial rate of 45Ca2+
release depended smoothly on InsP3 concentration, and InsP3 activated
release with moderate positive cooperativity. Preliminary experiments
performed at various free 40Ca2+ concentrations were consistent with a
bell-shaped 40Ca2+ dependence of 45Ca2+ release. In the range of micromolar
or higher free 40Ca2+ concentrations, the apparent inhibition of 45Ca2+
release was dependent on InsP3 concentration, and 45Ca2+ release for
intermediate InsP3 concentrations was transient; under selected conditions,
a second perfusion period, identical to the first one but separated from it
by a short recovery period, was found to allow renewed 45Ca2+ efflux. At
high Ca2+ concentration, fast reversible Ca(2+)-dependent desensitization
of the channel, and not heterogeneity, was therefore responsible for the
termination of InsP3-triggered 45Ca2+ efflux at submaximal concentrations
of InsP3. At lower Ca2+ concentrations, a large fraction of the apparent
activating effect of submicromolar 40Ca2+ concentrations on 45Ca2+ efflux
that we had observed in the preliminary experiments proved to be the
artifactual consequence of an inhibitory effect exerted by metal-free Ca2+
chelators on InsP3- dependent efflux at nanomolar 40Ca2+ concentrations.
1,2-Bis(2- aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid, EGTA, and fluo-3
were all effective inhibitors. When this inhibition was taken into account,
a rise in free 40Ca2+ concentration from 30 to 300 nM only weakly enhanced
45Ca2+ fluxes in the presence of a low concentration of InsP3. As a result,
submicromolar free 40Ca2+ appears to be only a poor activator of
InsP3-induced Ca2+ release under these experimental conditions.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
