Conditional fast expression and function of multimeric TRPV5 channels using Shield-1

1 Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen; and 2 Department of Metabolic and Endocrine Diseases and Netherlands Metabolomics Centre, UMC Utrecht, Utrecht, The Netherlands Submitted 7 August 2008 ; accepted in final form 6 October 2008 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. conditional protein expression; inducible system; ion channels; heteromultimerization Address for reprint requests and other correspondence: R. J. Bindels, Dept. of Physiology (286), Nijmegen Centre for Molecular Life Sciences, Radboud Univ. Nijmegen Medical Centre, Nijmegen 6500 HB, The Netherlands (e-mail: R.Bindels{at}ncmls.ru.nl )