Clustering of Ca²⁺ transients in interstitial cells of Cajal defines slow wave duration

Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca²⁺-activated Cl^- channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca²⁺ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca²⁺ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca²⁺ sensor, GCaMP3, in ICC. Ca²⁺ transients in ICC-MY display a complex firing pattern caused by localized Ca²⁺ release events arising from multiple sites in cell somata and processes. Ca²⁺ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca²⁺ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca²⁺ transients. Summation of CTCs results in relatively uniform Ca²⁺ responses from one slow wave to another. These Ca²⁺ transients are caused by Ca²⁺ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP₃ receptors. Reduced extracellular Ca²⁺ concentrations and T-type Ca²⁺ channel blockers decreased the number of firing sites and firing probability of Ca²⁺ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca²⁺ transients from multiple intracellular release sites. These events are organized into clusters by Ca²⁺ influx through T-type Ca²⁺ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves.