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

ABSTRACT: A long-standing goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships to understand how 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 7429 single-residue missense mutations 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 surface 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 known 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.

SUBMITTER: Coyote-Maestas W

PROVIDER: S-EPMC9273215 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM.

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

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

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