Hormone-evoked elementary Ca2+ signals are not stereotypic, but reflect activation of different size channel clusters and variable recruitment of channels within a cluster

Abstract

Previous studies of (InsP3)-evoked elementary Ca2+ events suggested a hierarchy of signals; fundamental events ("Ca2+ blips") arising from single InsP3 receptors (InsP3Rs), and intermediate events ("Ca2+ puffs") reflecting the coordinated opening of a cluster of InsP3Rs. The characteristics of such elementary Ca2+ release signals provide insights into the functional interaction and distribution of InsP3Rs in living cells. Therefore we investigated whether elementary Ca2+ signaling is truly represented by such stereotypic release events. A histogram of >900 events revealed a wide spread of signal amplitudes (20-600 nM; mean 216 +/- 4 nM; n = 206 cells), which cannot be explained by stochastic variation of a stereotypic Ca2+ release site. We identified elementary Ca2+ release sites with consistent amplitudes (<20% difference) and locations with variable amplitudes (approximately 500% difference). Importantly, within single cells, distinct sites displayed events with significantly different mean amplitudes. Additional determinants affecting the magnitude of elementary Ca2+ release were identified to be (i) hormone concentration, (ii) day-to-day variability, and (iii) a progressively decreasing Ca2+ release during prolonged stimulation. We therefore suggest that elementary Ca2+ events are not stereotypic, instead a continuum of signals can be achieved by either recruitment of entire clusters with different numbers of InsP3Rs or by a graded recruitment of InsP3Rs within a cluster.