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.
Highlights
Integral surface membrane proteins including ion channels, transporters, and receptors are vital to the survival and function of all cells
CHIP (C-terminus of the Hsp70interacting protein), is a U-box E3 ligase comprised of a catalytic domain that binds E2 and a tetratricopeptide repeats (TPR) targeting domain that binds Hsp70 (Connell et al, 2001; Murata et al, 2003; Zhang et al, 2005)
We have developed a toolset that enables post-translational ubiquitination of proteins in a specific and controllable manner, and applied these to study ubiquitin regulation of two distinct ion channels— KCNQ1 and CaV1.2
Summary
Integral surface membrane proteins including ion channels, transporters, and receptors are vital to the survival and function of all cells. Processes that control the surface abundance and composition of membrane proteins are critical determinants of cellular biology and physiology. Impaired surface trafficking of membrane proteins underlies diverse diseases ranging from cystic fibrosis to cardiac arrhythmias (Gelman and Kopito, 2002; Anderson et al, 2014), motivating a need to better understand fundamental mechanisms controlling membrane protein surface density. The surface repertoire of membrane proteins is regulated by multi-layered maturation and trafficking processes (MacGurn et al, 2012; Foot et al, 2017). Ubiquitination determines membrane protein functional expression by regulating multiple steps in the membrane protein lifecycle. Ubiquitin contains seven lysine residues (K6, K11, K27, K29, K33, K48, K63) that, together with its N-terminus (Met1), can serve as secondary attachment points to make diverse polyubiquitin chains with different structures and functions (Komander, 2009).
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