Different effects of glucose and potassium depolarization on the granule mobility in the submembrane space of insulin-secreting MIN6 cells
Background and aims: Physiologically, insulin secretion is initiated by an increase in the ambient glucose concentration. This involves a depolarization of the plasma membrane which is experimentally often mimicked by raising the K+ concentration in the medium. It has come into doubt, however, whether the stimulatory effect of a strong K+ depolarization, which produces a first phase-like insulin secretion, is based on the same mechanisms as the first phase of glucose-induced secretion.
Methods: Granules in the immediate vicinity of the plasma membrane were visualized by transient transfection of insulin-secreting MIN6 cells with an insulin-EGFP fusion protein and imaged by TIRF microscopy. The cells were continuously perifused with HEPES-buffered Krebs-Ringer medium (saturated with 95% O2 and 5% CO2) at 37.0 °C. The TIRF field had a calculated decay constant of 84nm. The image files (1 sequence = 200 images = mean duration of 25 seconds) were evaluated by an in-house written program (MATLAB 7.6.0) to achieve an observer-independent quantification. The free cytosolic Ca2+ concentration ([Ca2+]i) of MIN6 cells was measured with the Fura technique.
Results: Depending on the duration of their presence in the submembrane space, insulin granules were classified as either short-term (presence < 1 s) or long-term residents (presence for the entire image sequence ≥25 s). In a typical sequence ca. 80% of all the granules that were identified were short-term residents and 40 to 50% of the granules identified in the first image were still visible in the last image and were classified as long-term residents. Additionally, arrivals at and departures from the submembrane space were quantified, which represent the mobility in the z-direction, i.e. orthogonal to the plasma membrane. Raising the glucose concentration in the medium from 3 to 30 mM led to a marked increase of arrivals and departures within 2 min. During the next 7 min there was a further slight increase which was reversible upon wash-out of high glucose. A subsequent depolarization by 40 mM K+ raised again the rate of arrivals and departures, but this effect receded already during the K+ depolarization. Unexpectedly, this pattern was reflected more by the numbers of the long-term residents than by those of the short-term resident granules, which was practically constant. Specifically, the number of the long-term residents steadily decreased during glucose stimulation while there was a slight decrease followed by an increase during depolarization with 40 mM K+.
Conclusion: In contrast to current thinking K+ depolarization does not deplete the pool of docked (long-term resident) granules in the submembrane space, but diminishes it even less than glucose. Increased mobility may supply granules to release sites but may also serve to increase the turnover of docked granules, i.e. to “refresh” the pool of release-ready granules.