Characterization of Hyperactive Mutant forms of the Renal Potassium Channel, ROMK, a Protein Linked to Hypertension

Abstract

Hypertension affects one billion people worldwide and is the most common risk factor for cardiovascular disease. Despite the high heritability of hypertension, the relative contributions of known and as yet undiscovered genetic factors are not fully defined. However, amongst the regulators of hypertension are salt‐handling transporters in the kidney, one of which is the renal outer medullary K+ (ROMK) channel. Recent data from our labs indicate that unstable mutant forms of ROMK are prone to premature degradation, which give rise to Bartter syndrome Type II, a renal salt‐wasting disease. Intriguingly, heterozygous carriers of these same mutations are protected from hypertension. Therefore, we hypothesized that hyperactive, gain‐of‐function (GOF) mutations in ROMK that increase potassium flux will predispose individuals to hypertension. To test this hypothesis, we expressed ROMK mutants in yeast lacking its endogenous potassium transporters and then screened for potential GOF mutations that improve yeast growth on low K+. By utilizing both an unbiased genetic screen and a candidate‐based approach, we identified six GOF mutations, including one that exists in the NIH TOPMed human database. To assess how these mutations affect protein function and channel activity, we next performed cellular, biochemical, and electrophysiological assays in yeast, Xenopusoocytes, and human HEK293 cells. Data from these assays categorize the mutations into two main groups. In the first group, two mutations promote biosynthesis by stabilizing the proteins in the endoplasmic reticulum, thus promoting tetrameric channel assembly and increasing cell‐surface expression. In contrast, the second group reside in the PIP2‐binding pocket and significantly increase channel currents, yet protein synthesis is unaffected. Ongoing studies will elucidate how mutations in the PIP2 binding site allosterically affect channel function via patch‐clamp studies, and future efforts will be dedicated to developing murine models in which blood pressure regulation can be directly assessed. Ultimately, our findings will bring us one step closer to uncovering the complex genetics of hypertension as well as enhancing our understanding of how ROMK functions at the molecular level. Both efforts will aid in the development of new therapeutic strategies for hypertension.