Protein‐Protein Interaction Prediction Yields WNK‐SPAK/OSR1 Pathway as Regulator of Kir2.1 and Kir2.3 K+ Channels

Abstract

The WNK‐SPAK/OSR1 pathway is a key regulator of cellular ion transport and homeostasis, and has a role in physiological processes such as neurotransmission and blood pressure regulation. WNK kinases respond to changes in intracellular Cl−, osmolarity, and cell volume by activating the closely related kinases, SPAK and OSR1. The pathway mediates ion flux through Na+, K+, and Cl‐− cotransporters and ion channels via SPAK and OSR1‐dependent and independent mechanisms. SPAK and OSR1 use a protein interaction domain to bind linear R‐F‐x‐V/I sequence motifs in WNKs, cotransporters, and other proteins. Our goal was to identify novel SPAK and OSR1 interacting proteins by further defining the specificity determinants of the interaction, and predicting new interactions using the refined specificity profile. We used peptide arrays and biophysical measurements to define specificity, and used this information to search and rank motifs in the Uniprot database. All motifs not conserved in mice, not solvent exposed, and not present in the cytosol or nucleus were excluded. Nearly all of the previously validated interactions clustered within the top ten percent of our rankings and none were excluded, thus providing strong evidence that our ranking methodology was successful. Members of the inward rectifier K+ channel family were predicted to have a high probability of interacting with SPAK and OSR1 based on our rankings. We determined that OSR1 kinase activity activates two of the channels, Kir2.1 and Kir2.3, and activation is dependent on the presence of the binding motif. SiRNA knockdown of OSR1 and chemical inhibition of WNK kinases disrupted NaCl‐induced plasma membrane localization of Kir2.3. Our results suggest that activation of the WNK‐SPAK/OSR1 pathway enhances Kir2.1 and Kir2.3 channel activity by increasing plasma membrane localization of the channels. Our interaction predictions imply that additional inward rectifier K+ channel family members may be regulated in a similar manner. On a broader scale, our interaction predictions may help identify additional novel regulatory functions of the WNK‐SPAK/OSR1 pathway.Support or Funding InformationThis work was supported by National Institutes of Health GM53032 and Welch Foundation I‐1243 for M.H.C. Additional funding support to C.L.H.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.