Determinants of trafficking, conduction, and disease within a K⁺ channel revealed through multiparametric deep mutational scanning

Abstract

A longstanding goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships and delineate the mechanisms by which mutations cause disease. Deep Mutational Scanning (DMS) is a promising strategy to map how amino acids contribute to protein structure and function and to advance clinical variant interpretation. Here, we introduce 7,429 single residue missense mutation into the Inward Rectifier K+ channel Kir2.1 and determine how this affects folding, assembly, and trafficking, as well as regulation by allosteric ligands and ion conduction. Our data provide high-resolution information on a cotranslationally-folded biogenic unit, trafficking and quality control signals, and segregated roles of different structural elements in fold-stability and function. We show that Kir2.1 trafficking mutants are underrepresented in variant effect databases, which has implications for clinical practice. By comparing fitness scores with expert-reviewed variant effects, we can predict the pathogenicity of variants of unknown significance and disease mechanisms of know pathogenic mutations. Our study in Kir2.1 provides a blueprint for how multiparametric DMS can help us understand the mechanistic basis of genetic disorders and the structure-function relationships of proteins.

Deep Insertion, Deletion, and Missense Mutation Libraries for Exploring Protein Variation in Evolution, Disease, and Biology

“…In the helical (e.g., H109-L112 and V130-Q147) that determines potassium channel folding, and folding critical regions of the cytosolic C-terminal domain are completely immutable (e.g.,F203-V221, T276-D289, and S322-Y334) (Gajewski et al 2011; Fallen et al 2009). Overall, as in our previous DMS of Kir2.1 secondary structural elements are less mutable than unstructured regions (Coyote-Maestas et al 2022) (Figs 3D and 4).…”

“…Insertions are allowed which as well suggests that additional factors may mediate the evolutionary occurrence of indels, including mutational sampling and functional constraints. Pointing towards this, certain regions with known functional (but not trafficking) constraints, such as the CD loop, allow deletions in our data which are not observed during evolution (Coyote-Maestas et al 2022). Conversely, a cluster of indels which are extremely deleterious in our data occur within the onset of the H helix, which suggests these positions have become specialized for Kir2.1 trafficking or folding, and perhaps harbor unknown motifs.…”

“…The FCYENE is in the distal C terminus in non-folding critical regions meaning mutations here likely solely impact ER-export. In contrast, the SY motif interacts directly with the hydrophobic core so SY variants will additionally suffer dramatic folding deficits (Donghui Ma et al 2011; Coyote-Maestas et al 2022; Li et al 2016). With DIMPLE, we can confirm existing phenotypes within known trafficking motifs and in less understood proteins could discover new trafficking motifs and their boundaries.…”

Determinants of trafficking, conduction, and disease within a K+ channel revealed through multiparametric deep mutational scanning

“…The oligos are designed with unique barcodes for amplifying out a specific subpools library, Golden Gate-compatible BsmBI cut sites, and the mutation within a subsection of Kir2.1. OLS oligos were designed using the SPINE scripts on GitHub ( https://github.com/schmidt-lab/spine , Coyote-Maestas and Nedrud, 2022 ). These scripts design oligo libraries, primers for amplifying oligo-sublibraries, and inverse PCR primers for adding compatible cut sites to the Kir2.1 plasmid.…”

First Steps of Population Genomic Medicine in the Arrhythmia World: Pros and Cons

“…In conjunction with previous high-throughput functional studies by the same authors and others, 11 these data highlight how such an approach could comprehensively inform variant classification for both clinical genetic testing and population-level screening, with the goal to achieve saturation-level analysis of all possible missense variants for key arrhythmia genes. 12,13 However, it will be critical to extend these approaches to other genes currently less amenable to high-throughput functional assays. As demonstrated in this study, a focus on LMNA is particularly warranted on the basis of the apparent undercalling of pathogenic variants and the potentially severe phenotypes associated with variants in this gene.…”