Delivery of nonbiologically-compatible membrane protein constructs to mammalian cells for functional characterization.

Abstract

A hallmark of integral membrane protein biogenesis is the various cellular pathways that identify and tightly regulate protein fates. These quality control mechanisms identify misfolded from well-folded membrane proteins and target them for degradation or trafficking to the appropriate cellular membrane. A current limitation in membrane protein biophysics is that many engineered membrane proteins used in structural, molecular, and mechanistic studies; including mutations, truncations, and chimeras, do not satisfy cellular quality control requirements, limiting their ability for cellular characterization. Our recently developed membrane delivery method enables recombinantly expressed and purified membrane proteins to be delivered back to cells for functional and other characterization. Here we extend these methods to characterize the delivery and evaluate the function of a minimal TRPV1 ion channel by whole-cell patch-clamp electrophysiology and other techniques. This minimal construct is ∼35% of the full-length human TRPV1 receptor and does not satisfy cellular quality control, and is thereby excluded from trafficking to the plasma membrane. Delivery of minimal TRPV1 directly to the plasma membrane enables its characterization and shows that it retains polymodal activation by heat, protons, and capsaicin, analogously to the full-length TRPV1 protein. These outcomes are validated with other methods such as electron microscopy and NMR data, enabling a bridge between cellular and biophysical studies by bypassing cellular quality control and trafficking pathways.