Comparison of protein behavior between wild-type and G601S hERG in living cells by fluorescence correlation spectroscopy

Abstract

The human ether-a-go-go-related gene (hERG) protein is a cardiac potassium channel. Mutations in hERG can result in reductions in membrane channel current, cardiac repolarization, prolongation of QT intervals, and lethal arrhythmia. In the last decade, it has been found that some mutants of hERG involved in long QT syndrome exhibit intracellular protein trafficking defects, while other mutants sort to the membrane but cannot form functional channels. Due to the close relationship between intracellular trafficking and functional protein expression, we aimed to measure differences in protein behavior/motion between wild-type and mutant hERG by directly analyzing the fluorescence fluctuations of green fluorescent protein-labeled proteins using fluorescence correlation spectroscopy (FCS). Our data imply that FCS can be applied as a new diagnostic tool to assess whether the defect in a particular mutant channel protein involves aberrant intracellular trafficking.

Fig. 1

FCS monitors the random motion of fluorescently labeled molecules. These fluctuations provide information on the diffusion time of a particle and are directly dependent on the molecular size. For a large molecule, the fluorescence fluctuation is slow (a), whereas a small molecule makes rapid fluctuations (b). Consequently, any increase in the mass of a molecule (for example, as the result of an interaction with a second molecule) is readily detected as an increase in the particle’s diffusion time. An actual G(τ) plot curve of Rho6G is shown as an example (c)

Fig. 2

The localization of GFP-tagged wild-type hERG (a) and G601S (b) the day after transfection of their respective expression constructs. The left-hand panels show confocal-imaged, GFP-labeled G601S and WT hERG channels in HEK293 cells. The right-hand panels show merged Nomarski images and GFP fluorescence images. At 24 h after transfection, the protein localizations of WT hERG and G601S are indistinguishable. On day 2, approximately 48 h after transfection, WT hERG (c) was localized at the plasma membrane more than in the cytosol (arrow). In contrast, G601S was not trafficked through the cytosol to the plasma membrane (d) (arrow). All scale bars are 5 μm

Fig. 3

Comparison of diffusion coefficients between wild-type hERG and G601S in the cytosol on day 1 by FCS measurements. Both proteins contained two components of the diffusion coefficient. There was a statistically significant difference in the fast, D1 component between WT hERG (filled squares, dotted line circle) and G601S (unfilled circles, solid line circle). However, the slow, D2 component motions were statistically indistinguishable between WT hERG and G601S

Fig. 4

Comparisons of the D1 and D2 diffusion coefficients for wild-type hERG on day 1 (a) and day 2 (b) after transfection. The proportion of slowly diffusing particles was greater on day 2 than on day 1 (t test; p = 0.002). In the fluorescence images, GFP-WT hERG clearly localized at the plasma membrane (arrow). All scale bars are 5 μm