Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1: BTX tag or BTX incubation does not affect channel function. A) Representative inside-out patch-clamp recordings from COS cells cotransfected with BTX tag-SUR1 and WT Kir6.2 not pretreated (left) or pretreated for 30 min with TRITC-BTX at a concentration used in the imaging experiments (1:200) (right). In both cases, currents were readily inhibited by 1 mM ATP, similar to those seen in cells expressing non-tagged channels (48) (not shown). Recordings were made at −50 mV in symmetrical K-INT solution as described in Materials and Methods and inward currents are shown as upward deflections. B) Average current amplitude observed in patches from control and BTX-treated cells. The bar represents the mean ± SEM from 12 (control) or 10 (BTX-treated) cells. There is no statistically significant difference between the two groups by Student's t-test (p > 0.05).

Figure S2: WT BTX tag-Kir6.2 coexpressed with WT SUR1 shows characteristics similar to WT BTX tag-SUR1+WT Kir6.2. COSm6 cells were labeled with TRITC-BTX at 4°C and chased for 30 min at 37°C with the marker FITC-BSA included in the chase media. Cells transfected with WT BTX tag-Kir6.2/SUR1 displayed punctate TRITC-BTX staining (left panel, red, scale bar 10 µm) which colocalized with the fluid-phase marker FITC-BSA (middle panel, green), and thus confirming the intracellular nature of the channel staining (see merged picture and inset, right panel). Channel behavior was similar to WT BTX tag-SUR1/Kir6.2 channels.

All movies were compiled with METAMORPH software and play at 5 frames/second.

Movie S1: Movie corresponds to Figure 1A. A COS cell expressing BTX tag-SUR1 and WT Kir6.2 shows endocytosis of channels over a 30 min time interval. Images were taken every 15 seconds for 30 min (scale bar 5 µm).

Movie S2: Movie of INS-1 cells expressing BTX tag-SUR1 and WT Kir6.2 corresponding to Figure 1B. Images were taken every 15 seconds for 30 min (scale bar 5 µm). In both cases (Movies S1 and S2), internalization of KATP can be visually followed over time as fluorescent puncta budding from the plasma membrane and migrating to the cytosol.

Movie S3: Movie of a COS cell expressing SUR1 WT and BTX tag-Kir6.2. Images were taken every 15 seconds for 15 min. The BTX tag can be fused to either KATP channel subunit with no difference in internalization behavior (compare to Movie S1A, scale bar 10 µm).

Movie S4: 30 min movie of COS cells expressing BTX tag-SUR1 and Kir6.2 Y330C corresponding to Figure 2A. Internalization of BTX tag-SUR1/Kir6.2 Y330C is similar to wild-type channels (scale bar 5 µm).

Movie S5: Movie of COS cells expressing BTX tag-SUR1RKR[RIGHTWARDS ARROW]AAA only. SUR1 internalizes independently from Kir6.2 when the ER-retention signal is mutated (30 min movie, scale bar 10 µm). This movie corresponds to Figure 6A, top as control for the dynasore-treated cell.

Movie S6: Movie of COS cells expressing BTX tag-SUR1RKR[RIGHTWARDS ARROW]AAA with the drug dynasore in the imaging media. SUR1RKR[RIGHTWARDS ARROW]AAA internalization is greatly reduced when dynasore, an inhibitor of dynamin, was present (30-min movie, scale bar 10 µm).

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