MONITORING DYNAMIC CHANGES IN FREE CA2-RETICULUM OF INTACT-CELLS( CONCENTRATION IN THE ENDOPLASMIC)

Direct monitoring of the free Ca2+ concentration in the lumen of the e ndoplasmic reticulum (ER) is an important but still unsolved experimen tal problem, We have shown that a Ca2+-sensitive photoprotein, aequori n, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences, By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show he re that, after emptying the ER, Ca2+ is rapidly re-accumulated up to c oncentrations of >100 mu M, thus consuming most of the reporter photop rotein. An estimate of the steady-state Ca2+ concentration was obtaine d using Sr2+, a well-known Ca2+ surrogate which elicits a significantl y slower rate of aequorin consumption, Under conditions in which the r ate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+](er)) wa s similar to 2 mM, Receptor stimulation causes, in a few seconds, a 3- fold decrease of the [Sr2+](er), whereas specific inhibition of the ER Ca2+ ATPase leads to an similar to 10-fold drop in a few minutes.