Fig. 1. Zinc transport in Min6 cells. (a) Min6 insulinoma cell line, grown on coverslips, was loaded with Fura-2 AM () and changes in fluorescence monitored. A rise in fluorescence resulting from cation permeation following depolarization by high-K⁺ (50 mM KCl replacing NaCl) Ca²⁺-free Ringer’s solution is apparent. The cell-permeable Zn²⁺ chelator, TPEN, reduced the fluorescence to baseline levels, indicating that it was a Zn²⁺-dependent signal. (b) Cells were loaded with Fura-2 and Zn²⁺ (75 μM) permeation was monitored in Ca²⁺-free Ringer’s solution. The application of 100 μM verapamil completely blocked Zn²⁺ influx. (c) Zn²⁺ influx was monitored in cells loaded with the Zn²⁺-sensitive dye (Newport green, ) in the presence or absence of the LTCC blocker, nifedipine (in Ca²⁺-free Ringer’s). Application of nifedipine completely blocked Zn²⁺ influx. Our results indicate that the Zn²⁺ influx was mediated by LTCC.

Fig. 2. Zinc, at physiological concentrations, permeates via the L-type Ca²⁺ channels into Min6 cells. (a) Cells were depolarized in the presence of the indicated Zn²⁺ concentrations, in Ca²⁺-free Ringer’s. The calibrated pZn (−log₁₀ [free Zn²⁺]) values corresponding to the concentrations added are: 6.05, 5.73, 5.43, 5.13, 4.82, 4.43, 4.28, and 4.12. Note that when Zn²⁺ was added prior to depolarization, no change in the fluorescence signal was observed. Zinc influx was observed at low zinc concentration (less than 5 μM). (b) The initial rate of the fluorescence response was calculated and is plotted against the applied Zn²⁺ concentration. Fitting the data to the Michaelis–Menten equation yielded a K_m for Zn²⁺ of 37 ± 5μM (free Zn²⁺).

Fig. 3. Zn²⁺ is retained in the cytosol of Min6 cells, while Ca²⁺ is rapidly removed. (a) Ca²⁺ influx was monitored in Zn²⁺-free or Zn²⁺-containing Ringer’s solution using cells loaded with Fura-2, which is sensitive to both Zn²⁺ and Ca²⁺. Following depolarization in Ca²⁺-containing Ringer’s, the fluorescent signal rise is similar in the presence or absence of Zn²⁺. Subsequently, the cells were washed, and a decrease in fluorescence observed. The rate of fluorescence decrease in the marked region is shown in the inset and is significantly smaller in the presence of Zn²⁺ (p < 0.02). In the Zn²⁺-containing Ringers’ solution, the fluorescent signal decreased to an elevated plateau which was eliminated only by the application of TPEN. (b) Zinc influx was monitored in cells loaded with the Zn²⁺-specific dye, Newport green, in the absence or presence of extracellular Ca²⁺ in the Ringer’s solution. The similar rates of increase in fluorescence suggest that Zn²⁺ influx through LTCC is maintained in the presence of physiological concentrations of Ca²⁺.

Fig. 4. Sequestration of Zn²⁺ into vesicles in Min6 cells. (a) Min6 cells, loaded with Fura-2, were depolarized in the presence of Zn²⁺ (free Zn²⁺ concentrations 3.7 or 75 μM) for 1 or 5 min (in Ca²⁺-free Ringer’s), and the fluorescent signal monitored. A rise in the fluorescent signal, indicating Zn²⁺ permeation, is apparent throughout the duration of the depolarization. Zn²⁺ accumulation in cells exposed for 5 min to 3.7 μM Zn²⁺, was approximately threefold larger than in cells exposed to the same concentration for only 1 min. Fluorescence in cells exposed for 5 min to 3.7 μM Zn²⁺ or for 1 min to 75 μM Zn²⁺ remained at an elevated level following a small initial decrease. (b) Cells were exposed for 1 min to 3.7 or 75 μM Zn²⁺ (in Ca²⁺-free Ringer’s) and then loaded with ZP1. The right panel shows the fluorescent image while in the left panel, the same image is overlaid on a brightfield image of the cells, images were acquired using a 100× objective. In cells loaded with low concentrations of Zn²⁺, fluorescent signal is localized to vesicle-like structures within the cells, while the high Zn²⁺ concentration resulted in diffuse fluorescence within the cytosol.

Fig. 5. Permeation of Zn²⁺ via the LTCC into Min6 cells induces β-cell death. (a) Min6 cells were depolarized with 50 mM KCl-Ringer’s containing 113 μM Zn²⁺ for 1–10 min (in Ca²⁺-free Ringer’s), and MTT assay was performed 24 h later. *p < 0.01, **p  0.005, versus 1 min exposure to Zn²⁺. (b) The same experimental paradigm was applied in the presence of the LTCC blocker, nimodipine 1 μM, or was followed by application of TPEN (100 μM). Cell viability was similar in cells treated with the LTCC blocker, the Zn²⁺ chelator or in the non-treated controls. ***p < 0.0005.