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Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca

Electrical activity and oscillations of cytosolic Ca2+ concentrations ([Ca2+]i) that trigger insulin release in response to glucose are key functions of pancreatic β cells. Although oscillatory Ca2+ signals have been intensively studied in β cells, their lower frequency did not match that of electrical activity. In addition, the measured peak [Ca2+]i did not reach levels that are typically required by synaptotagmins to elicit the release of insulin-containing vesicles in live-cell experiments. We therefore sought to resolve the Ca2+ dynamics in the subplasmalemmal microdomain that is critical for triggering fast exocytosis. Applying total internal reflection fluorescence (TIRF) microscopy in insulin-producing INS-1E and primary mouse β cells, we resolved extraordinary fast trains of Ca2+ spiking (frequency > 3 s−1) in response to glucose exposure. Using a low-affinity [Ca2+]i indicator dye, we provide experimental evidence that Ca2+ spikes reach low micromolar apparent concentrations in the vicinity of the plasma membrane. Analysis of Ca2+ spikes evoked by repeated depolarization for 10 ms closely matched the Ca2+ dynamics observed upon glucose application. To our knowledge, this is the first study that experimentally demonstrates Ca2+ spikes in β cells with velocities that resemble those of bursting or continuously appearing trains of action potentials (APs) in non-patched cells.