Solitary calcium spike dependent on calmodulin and plasma membrane Ca2+ pump.

Resealed human red cell ghosts were loaded with Fura-2, ATP, Mg2+, and either calmodulin (CaM) or, to prevent CaM activation of the Ca2+ pump, a synthetic peptide that antagonized endogenous CaM (an analogue of the CaM binding domain of protein kinase II, referred to as 'antiCaM'). The ghosts reduced the cytosolic concentration of ionized calcium (Ca2+]i) to 193 +/- 60 nM (SD, n = 15) in a medium containing 1 mM Ca2+ and to 30 +/- 27 nM (SD, n = 62) in a medium without Ca2+ addition. Without ATP, i.e. no fuelling of the Ca2+ pump, the Ca2+]i remained high (approx. 5 microM or higher). The simultaneous addition of the ionophore A23187 and Ca2+ rapidly increased the Ca2+ influx, which in the CaM loaded ghosts caused a solitary spike of Ca2+]i, reaching maximum around 2 microM within 24 +/- 6 s (SD, n = 40). On the contrary, in the ghosts loaded with antiCaM, the addition of A23187 with Ca2+ raised Ca2+]i during the first 2 min to a high level (2-4 microM) with no preceding spike. Pre-incubation of CaM-ghosts with Ca2+ diminished the height of the Ca2+ spike, and treatment with trypsin even removed the Ca2+ spike. The trypsin treatment activated the Ca2+ pump prior to the rise of Ca2+]i, making the time-consuming CaM activation unnecessary. In conclusion, the Ca2+ spiking is dependent on a delayed CaM activation of the plasma membrane Ca2+ pump in response to a rapid increase of Ca2+ influx.