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.
Dept. of Physiology (286), Nijmegen Centre for Molecular Life Sciences, Radboud Univ. Nijmegen Medical Centre, Nijmegen 6500 HB, The Netherlands.
Joost P H
JP
Schoeber
Stan F J
SF
van de Graaf
Kyu Pil
KP
Lee
Hanneke G M
HG
Wittgen
Joost G J
JG
Hoenderop
René J M
RJ
Bindels
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