Sculpting ion channel functional expression with engineered ubiquitin ligases

Abstract

The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K+ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr t1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels.

Designer genetically encoded voltage‐dependent calcium channel inhibitors inspired by RGK GTPases

“…Indeed, we have previously shown that the nanobody‐based targeted ubiquitination approach can be used to inhibit KCNQ1 channels by eliminating them from the cell surface (Kanner et al . 2017).…”

“…2012; Kanner et al . 2017). In heterologous cells, nb.F3‐Nedd4L decreased the surface density of reconstituted Ca V 2.2 and Ca V 1.2 channels without enhancing the degradation of the pore‐forming α 1B and α 1C subunits, respectively (Fig.…”

Proteome editing using engineered proteins that hijack cellular quality control machinery

“…A distinct advantage of uAbs is their highly modular architecture which enables target selection to be rewired by simply swapping the synthetic protein‐binding domain. For example, targeted proteolysis has been achieved for a diverse array of protein substrates including eukaryotic proteins (ASC, HRAS/KRAS, Lck, and SHP2,), intraneuronal bacterial proteins ( Clostridium botulinum neurotoxin [BoNT] proteases), fluorescent proteins (FPs), and dozens of FP‐tagged proteins that range in size from 27 to 179 kDa and localize in different subcellular compartments including the cytoplasm, nucleus, and cell membrane . Moreover, by incorporating synthetic binding proteins that recognize particular protein states (e.g., active vs. inactive conformation, mutant vs. wild‐type, posttranslationally modified, and so forth), it becomes possible to deplete certain protein subpopulations while sparing others .…”

A genetically-encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

“…However, they also offer a generic means of targeting in live cells the huge variety of available tagged proteins and the many emerging examples of endogenous proteins tagged with FPs by gene editing. GFP-targeting nanobodies have been used for applications such as targeted proteosomal degradation [23, 24] and relocation of proteins in cells [25], but these and other applications are less developed for RFP-targeting nanobodies.…”