Underdominant KCC3b R31I association with blood sodium concentration in domestic sheep suggests role in oligomer function

Authors: White, Stephen; OLIVEIRA, RYAN - Washington State University; Mousel, Michelle; GONZALEZ, MICHAEL - University Of Pennsylvania; Highland, Margaret; HERNDON, MARIA - Washington State University; Taylor, Joshua - Bret; Knowles Jr, Donald

Submitted to: Animal Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/13/2017
Publication Date: 10/1/2017
Citation: White, S.N., Oliveira, R.D., Mousel, M.R., Gonzalez, M.V., Highland, M.A., Herndon, M.K., Taylor, J.B., Knowles Jr, D.P. 2017. Underdominant KCC3b R31I association with blood sodium concentration in domestic sheep suggests role in oligomer function. Animal Genetics. 48(5):626-627. https://doi.org/10.1111/age.12585.
DOI: https://doi.org/10.1111/age.12585

Interpretive Summary: The potassium chloride co-transporter 3 (KCC3) is an ion pump that is strongly related to hypertension. There are two versions of the gene activated at different starting points: KCC3a is widely expressed throughout the body, and KCC3b is highly expressed in at a specific point in the kidney. We used 307 sheep to demonstrate an association between a charged amino acid substitution in the kidney expressed KCC3b (designated R31I) and blood sodium concentration. This is the first report of a mutation in potassium transporter KCC3 associated with blood sodium. The association showed an underdominant pattern where sheep with two copies of either the R amino acid or the L amino acid had similar sodium concentrations, but sheep with one copy of each amino acid had significantly lower blood sodium. Underdominance is an unusual pattern, and it often occurs when a protein that functions as a homodimer (two copies of the protein work together to perform a function) has a mutation that impairs ability of protein copies to work together. KC3 is known to function as a homodimer, and this result suggests the R31I substitution may interfere with KCC3 dimerization. This work provides important information about function of mutations in a poorly characterized part of an important hypertension gene, and suggests future work to understand how a potassium transporter may influence sodium. It also suggests that sheep with this mutation could be a biomedical model to investigate such questions.

Technical Abstract: KCC3 and KCC1 are potassium chloride transporters with partially overlapping function, and KCC3 knockout mice exhibit hypertension. Two KCC3 isoforms differ by alternate promoters and first coding exons: KCC3a is widely expressed, and KCC3b is highly expressed in kidney proximal convoluted tubule. We genotyped KCC3 and KCC1 amino acid substitutions using Taqman assays in 307 Suffolk, Rambouillet, Polypay, and Columbia sheep. Whole blood sodium and potassium concentrations were determined by atomic absorbance spectrometry. Association was determined by mixed models in SAS 9.2 (SAS Institute, Cary, NC) with breed and genotype for the variant of interest as independent fixed effects, and sire nested within breed treated as random. Preliminary testing showed age in years was not related to sodium or potassium concentration (P>0.05), so age was not included in the models. No KCC3a or KCC1 substitutions were associated with blood potassium or sodium (all P>0.05). KCC3b R31I is a charged substitution located in a conserved motif and while not associated with blood potassium (P>0.05), it was associated with blood sodium in an underdominant manner where blood sodium was significantly higher in both homozygotes than in heterozygotes (P<=0.05). The sodium association is interesting because: 1) KCC3 is only known to transport potassium and chloride, not sodium, and 2) KCC3a interacts with the sodium-potassium pump, whereas KCC3b does not. Possible mechanisms include: A) R31I alteration of KCC3b sodium affinity, B) KCC3b oligomer formation with sodium transporters, or C) indirect influence, e.g. by impairing sodium-potassium pump efficiency through potassium availability. Regardless of specific mechanism, the underdominant pattern suggests allelic incompatibility such as dimer impairment. Since KCC3 functions as a homodimer, R31I may interfere with KCC3b dimer function in regulating sodium concentration. To our knowledge, this is the first report of a KCC3 variant associated with blood sodium, and these data suggest further study of coordinated function between KCC3 and sodium transport.