A novel, radiolabel-free pulse chase strategy to study Kir3 channel ontogeny.
Kir3 channels are essential regulators of cellular excitability, maintaining cells at resting membrane potentials. While much research has been dedicated to elucidating the mechanisms regulating Kir3 channel gating, little is known regarding the channel's early associations with signaling partners, its stability at the plasma membrane or mechanisms regulating its internalization and degradation. To address these issues we have established an inducible Kir3.1 cell line that allows monitoring of a discrete "pulse" of channel as it progresses along the biosynthetic pathway. Using this system, we have been able to track Kir3 maturation and the influence of partner subunits on Kir3 lifetime and stability. Of note, we show that Kir3.1, in the absence of trafficking partner subunits, can exit the endoplasmic reticulum (ER) and reach the Golgi (though not the plasma membrane), and that expression of Kir3.3 subunits drastically reduced levels of Kir3.1 in the cell. We also show that interfering with trafficking from the ER to Golgi has a pronounced inhibitory effect on Kir3.1-Kir3.2 interactions, suggesting that this complex is stabilized either en route to the Golgi or in the Golgi itself. Finally, we showed that the Kir3 channel can reach the cell surface as early as 6 h post-induction and that removal of cell surface-localized channel occurs within 48 h. This system can be adapted to study the life cycle of any cellular protein without the confounds associated with radioactive labeling or the complications noted with expressing supraphysiological levels of proteins.
Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada.
Peter
P
Zylbergold
Rory
R
Sleno
Terence E
TE
Hébert
D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D008564:Membrane Potentials / Q000235:genetics*; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D015398:Signal Transduction
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D021122:Protein Subunits