Controlling ion channel trafficking by targeted ubiquitination and deubiquitination

Plasma membrane-localized ion channels are essential for diverse physiological processes such as neurotransmission, muscle contraction, and osmotic homeostasis. The surface density of such ion channels is a major determinant of their function, and tuning this variable is a powerful way to regulate physiology. Dysregulation of ion channel surface density due to inherited or de novo mutations underlies many serious diseases, and molecules that can correct trafficking deficits are potential therapeutics and useful research tools. We have developed targeted ubiquitination and deubiquitination approaches that enable selective posttranslational down- or up-regulation, respectively, of desired ion channels. The method employs bivalent molecules comprised of an ion-channel-targeted nanobody fused to catalytic domains of either an E3 ubiquitin ligase or a deubiquitinase. Here, we use two examples to provide detailed protocols that illustrate the utility of the approach-rescued surface expression of a trafficking-deficient mutant K_V7.1 (KCNQ1) channel that causes long QT syndrome, and selective elimination of the Ca_V2.2 voltage-gated calcium channel from the plasma membrane using targeted ubiquitination. Important aspects of the approach include having a robust assay to measure ion channel surface density and generating nanobody binders to cytosolic domains or subunits of targeted ion channels. Accordingly, we also review available methods for determining ion channel surface density and nanobody selection.