Abstract
The renal outer medullary potassium (ROMK) channel is essential for potassium transport in the kidney, and its dysfunction is associated with a salt-wasting disorder known as Bartter syndrome. Despite its physiological significance, we lack a mechanistic understanding of the molecular defects in ROMK underlying most Bartter syndrome-associated mutations. To this end, we employed a ROMK-dependent yeast growth assay and tested single amino acid variants selected by a series of computational tools representative of different approaches to predict each variants’ pathogenicity. In one approach, we used in silico saturation mutagenesis, i.e. the scanning of all possible single amino acid substitutions at all sequence positions to estimate their impact on function, and then employed a new machine learning classifier known as Rhapsody. We also used two additional tools, EVmutation and Polyphen-2, which permitted us to make consensus predictions on the pathogenicity of single amino acid variants in ROMK. Experimental tests performed for selected mutants in different classes validated the vast majority of our predictions and provided insights into variants implicated in ROMK dysfunction. On a broader scope, our analysis suggests that consolidation of data from complementary computational approaches provides an improved and facile method to predict the severity of an amino acid substitution and may help accelerate the identification of disease-causing mutations in any protein.
Highlights
The Renal Outer Medullary Potassium (K) Channel, ROMK, is a member of the Kir family of inwardly rectifying potassium transporters and resides most prominently in two regions of the kidney, the thick ascending loop of Henle and the cortical collecting duct [1]
While predictive algorithms are available for this purpose, a comparative analysis of recently developed algorithms has not been adequately performed, nor is it clear whether combining algorithms would improve predictive power
We focus on ROMK single amino acid variants (SAVs) motivated by: (i) their potential link to a catastrophic disease, type 2 Bartter syndrome, for which there is no cure, (ii) the fact that the effects of most disease-causing mutations on ROMK function are ill-defined, and (iii) the lack of knowledge on the consequences of numerous SAVs in ROMK, which have been reported in the human genome database
Summary
The Renal Outer Medullary Potassium (K) Channel, ROMK, is a member of the Kir family of inwardly rectifying potassium transporters and resides most prominently in two regions of the kidney, the thick ascending loop of Henle and the cortical collecting duct [1]. Members of the seven families of Kir channels are found in every major organ and can be gated by voltage or cyclic nucleotides, whereas others, like ROMK, are constitutively active [2]. The levels and activity of ROMK are instead controlled by biosynthesis, transport, or retention at the plasma membrane, as well as by pH, PIP2, and protein tyrosine kinases. The active plasma membrane resident ROMK species is formed from four identical ~45 kDa polypeptides. Like other membrane proteins that reside at the plasma membrane, ROMK is initially synthesized on endoplasmic reticulum (ER)-associated ribosomes. ROMK monomer folding and tetramer assembly most likely take place in the ER, which are essential for subsequent trafficking to the cell surface
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