Molecular basis and physiological functions of dynamic Ca2+ signalling in smooth muscle cells.

We have visualized Ca2+ signals in smooth muscle cells mediated by the release of Ca2+ from intracellular Ca2+ stores and studied their underlying molecular basis. Ca2+ signals in smooth muscle cells within intact arterial tissues show diverse spatiotemporal patterns: Ca2+ waves and oscillations were induced by agonist stimulation or by sympathetic nerve stimulation. We also found spontaneous Ca2+ oscillations with low amplitudes (Ca2+ ripples) that were observed in the absence of extrinsic stimulation. These dynamic spatiotemporal patterns were generated by Ca2+ release via the inositol-1,4,5-trisphosphate (InsP3) receptor (InsP3R). We then studied the molecular basis of such complex Ca2+ signalling patterns. The activity of InsP3R is regulated by the cytoplasmic Ca2+ concentration. The sensitivity of InsP3R to Ca2+ provides feedback regulation of the Ca2+ release, which may be important for the generation of Ca2+ signalling patterns. A series of site-specific mutagenesis experiments in type 1 InsP3R allowed us to identify glutamate at position 2100 as the Ca2+ sensor. Substitution of the amino acid by aspartic acid resulted in a 10-fold decrease in Ca2+ sensitivity. In cells expressing the mutant InsP3R, Ca2+ release spikes and oscillations were inhibited, indicating the role of the Ca2+ sensitivity of InsP3R in the generation of spatiotemporal patterns of Ca2+ signals.