Submitted on August 7, 2008
Revised on October 3, 2008
Accepted on October 6, 2008
Conditional fast expression and function of multimeric TRPV5 channels using Shield-1
Joost P.H. Schoeber1, Stan F.J. van de Graaf2, Kyu Pil Lee1, Hanneke G.M. Wittgen1, Joost G.J. Hoenderop1, and Rene J.M. Bindels1*
1 Radboud University Nijmegen Medical Centre
2 UMC Utrecht
* To whom correspondence should be addressed. E-mail: r.bindels{at}ncmls.ru.nl.
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 co-degraded upon washout of Shield-1 - providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.
