Abstract

Hormones and neurotransmitters that act through inositol 1,4,5-trisphosphate (IP₃) can induce oscillations of cytosolic Ca²⁺ ([Ca²⁺]_c), which render dynamic regulation of intracellular targets. Imaging of fluorescent Ca²⁺ indicators located within intracellular Ca²⁺ stores was used to monitor IP₃ receptor channel (IP₃R) function and to demonstrate that IP₃-dependent oscillations of Ca²⁺ release and re-uptake can be reproduced in single permeabilized hepatocytes. This system was used to define the minimum essential components of the oscillation mechanism. With IP₃ clamped at a submaximal concentration, coordinated cycles of IP₃R activation and subsequent inactivation were observed in each cell. Cycling between these states was dependent on feedback effects of released Ca²⁺ and the ensuing [Ca²⁺]_c increase, but did not require Ca²⁺ re-accumulation. [Ca²⁺]_c can act at distinct stimulatory and inhibitory sites on the IP₃R, but whereas the Ca²⁺ release phase was driven by a Ca²⁺-induced increase in IP₃ sensitivity, Ca²⁺ release could be terminated by intrinsic inactivation after IP₃ bound to the Ca²⁺-sensitized IP₃R without occupation of the inhibitory Ca²⁺-binding site. These findings were confirmed using Sr²⁺, which only interacts with the stimulatory site. Moreover, vasopressin induced Sr²⁺ oscillations in intact cells in which intracellular Ca²⁺ was completely replaced with Sr²⁺. Thus, [Ca²⁺]_c oscillations can be driven by a coupled process of Ca²⁺-induced activation and obligatory intrinsic inactivation of the Ca²⁺-sensitized state of the IP₃R, without a requirement for occupation of the inhibitory Ca²⁺-binding site.