Function recovery after chemobleaching (FRAC): evidence for activity silent membrane receptors on cell surface

Abstract

Membrane proteins represent approximately 30% of the proteome of both prokaryotes and eukaryotes. Unique to cell surface receptors is their biogenesis pathway, which involves vesicular trafficking from the endoplasmic reticulum through the Golgi apparatus and to the cell surface. Increasing evidence suggests specific regulation of biogenesis for different membrane receptors, hence affecting their surface expression. We report the development of a pulse-chase assay to monitor function recovery after chemobleaching (FRAC) to probe the transit time of the Kir2.1 K+ channel to reach the cell surface. Our results reveal that the channel activity is contributed by a small fraction of channel protein, providing evidence of activity-silent "sleeping" molecules on the cell surface. This method distinguishes molecular density from functional density, and the assay strategy is generally applicable to other membrane receptors. The ability of the reported method to access the biogenesis pathways in a high-throughput manner facilitates the identification and evaluation of molecules affecting receptor trafficking.

Fig. 1.

A schematic diagram outlining the FRAC assay.

Fig. 2.

Rb⁺ flux assays of HEK 293 cells transiently and stably expressing Kir2.1Y channel. (a and b) In Rb⁺ influx (a) and efflux (b), 293 cells were transiently transfected with Kir2.1Y expression vector (▪)or empty vector (•). Data obtained with 2.5 mM MTSET treatments are shown as □ or ○. (c) Rb⁺ efflux of 293 cells stably expressing Kir2.1Y channel with (□) or without (▪) MTSET treatment were measured. (d and e) Determination of MTSET treatment conditions. (d) Rb⁺ efflux (%) of 293 cells stably expressing Kir2.1Y channel (filled bars) and mock 293 cells (open bars) were quantified after being treated in triplicates with 5 mM MTSET for 0, 2.5, 5 and 10 min at room temperature. (e)Rb⁺ efflux (%) of 293 cells stably expressing Kir2.1Y channel (filled bars) and mock 293 cells (open bars) were quantified after being treated in duplicate with 0, 0.5, 1, 2.5 and 5 mM MTSET for 5 min at room temperature.

Fig. 3.

Detection of Kir2.1Y and Kir2.1 channel functional surface expression. (a) K⁺ channel activity measured by Rb⁺ efflux assayed at the indicated time periods. Results are for 293 cells stably expressing either Kir2.1 (Upper)or Kir2.1Y (Lower) channel after MTSET treatment (□), Rb⁺ efflux of 293 cells stably expressing Kir2.1 channel without MTSET treatment (▪) and mock 293 cells treated with (○) or without (•) MTSET. (b) Flow cytometry analyses of 293 cells stably expressing Kir2.1Y channel with MTSET treatment at different time points (0, 3, and 6 h) before staining with anti-HA antibody to quantify the surface expression of Kir2.1Y. FL, fluorescence intensity in a logarithmic scale. Events refers to the number of cells.

Fig. 4.

Temperature-dependent activity recovery of Kir2.1Y channel determined by Rb⁺ efflux. (a–c) Activity recovery of Kir2.1Y stable cell line after MTSET treatment (□), Rb⁺ efflux of Kir2.1Y stable cell line without MTSET treatment (▪), and Rb⁺ efflux of mock 293 cells after MTSET treatment (○)or without MTSET treatment (•). The recovery experiments were carried out at 15°C (a), 22°C (b), and 30°C (c). All measurements were performed in triplicate as described in Methods. (d) Normalized recovery rates from 0 to 5 h at 15°C (▪), 22°C (□), 30°C (▴), and 37°C (⋄) (from Fig. 3a). The Rb⁺ efflux of Kir2.1Y without MTSET treatment was set to 100%.

Fig. 5.

BFA effect on activity recovery of Kir2.1Y channel. (a) We incubated 293 cells stably expressing Kir2.1Y (hatched bars) and mock 293 cells (open bars) with 1 μM BFA at 37°C for 3 h after a 5-min MTSET treatment or without MTSET treatment. In control samples, neither MTSET nor BFA were added. Each column is the average of two samples. (b) Dose-dependent effect by BFA treatment. Results show 293 cells stably expressing Kir2.1Y that were incubated with BFA only (▪), cells stably expressing Kir2.1Y that were treated with MTSET before incubation with BFA (□), mock 293 cells incubated with BFA only (•), and mock 293 cells treated with MTSET before incubation with BFA (○). All experiments were performed in duplicate with 3-h incubation. The Rb⁺ efflux (%) is plotted against different concentrations of BFA at 37°C before Rb⁺ efflux assay.