Conditional fast expression and function of multimeric TRPV5 channels using Shield-1.
A recently described novel controllable method to regulate protein expression is based on a mutated FK506-binding protein-12 (mtFKBP) that is unstable and rapidly degraded in mammalian cells. This instability can be conferred to other proteins directly fused to mtFKBP. Binding of a synthetic cell-permeant ligand (Shield-1) to mtFKBP reverses the instability, allowing conditional expression of mtFKBP-fused proteins. We adapted this strategy to study multimeric plasma membrane proteins using the ion channel TRPV5 as model protein. mtFKBP-TRPV5 forms functional ion channels and its expression can be controlled in a time- and dose-dependent fashion using Shield-1. Moreover, in the presence of Shield-1, mtFKBP-TRPV5 formed heteromultimeric channels with untagged TRPV5, which were codegraded upon washout of Shield-1, providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.
Schoeber
Joost P H
JP
Dept. of Physiology (286), Nijmegen Centre for Molecular Life Sciences, Radboud Univ. Nijmegen Medical Centre, Nijmegen 6500 HB, The Netherlands.
van de Graaf
Stan F J
SF
Lee
Kyu Pil
KP
Wittgen
Hanneke G M
HG
Hoenderop
Joost G J
JG
Bindels
René J M
RJ
D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008024:Ligands; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D054852:Small Molecule Libraries / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism*
D015220:Calcium Channels; D008024:Ligands; D054852:Small Molecule Libraries; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C422827:TRPV6 protein, human; D022061:Tacrolimus Binding Protein 1A