PROJECT SUMMARY/ABSTRACT Regulated transport of Na+ and K+ in the kidney tubules is central to blood pressure regulation and fluid and electrolyte homeostasis. The mTORC2-SGK1-ENaC axis is a well-established component of this regulatory machinery, however, key mechanistic features remain poorly characterized, and in vivo data is limited. We have used in vivo and in vitro approaches to identify novel features of this signaling system, particularly its role in K+ homeostasis. Our recent data suggest that: 1) K+ acts through WNK kinases to activate mTORC2; 2) tubule cell mTORC2 is central to the rapid response to a K+ load in mice; 3) Structural features of mTORC2, elucidated using cryo-EM, play an essential role in mTORC2 activity and specificity. To explore these hypotheses, we will: Aim 1: Assess the temporal sequence of responses to K+ in mice with tubule-specific knockout of mTORC2. We have generated a rapidly inducible kidney tubule-specific Rictor KO (TRKO) mouse model. Preliminary data show that these mice fail to respond to KCl normally; differences between WT and KO mice manifest in <3h following KCl gavage, and are striking by 48 h of high K diet. To characterize these mice, we will determine: (A) Time course of response to acute KCl load. Mice will be treated with an oral K+ load and urinary and plasma parameters will be assessed. Kidneys will be harvested and assessed for tissue signaling parameters and ion transporter expression and modification. ENaC, ROMK, and BK channel activities will be measured using patch clamp. Cytoplasm from patched cells will be captured and mRNA subjected to RNA-seq to identify single-cell gene expression patterns. (B) Time course of gene deletion: Mice will be adapted to a high K+ diet prior to initiating gene deletion. Balance experiments will be performed on adrenal-intact and ADX + aldo mice at time points prior to and during emergence of phenotype. Patch clamp will be performed and cytoplasmic RNA harvested and analyzed as in (A). (C) Evaluate PKC role and identify novel targets. We will identify key PKC substrates using LC/MS, and functionally characterize them in vivo and in cultured cells. Aim 2: mTORC2 molecular signaling mechanisms and specificity: structural features and effects of WNK kinases. We will establish the cellular and molecular features that underlie mTORC2 regulation of SGK1 using cultured cells and cryo-EM. (A) Investigate WNK1 and WNK4 as scaffolds that promote mTORC2-dependent phosphorylation of SGK1 in a K+-dependent fashion. We will examine effects of WNK kinases on physical interactions and phosphorylation of SGK1, and functional effects on ENaC and ROMK in cultured mpkCCD and HEK-293 cells. (B) Cryo-EM structural analysis of mTORC2 core complex, and with SGK1 and WNK1. mTORC2 core components will be subjected to cryo-EM in the presence and absence of SGK1 and WNK kinases. Aim 3: Assess the roles of WNK1 and WNK4 and functional interactions with mTORC2 in vivo using inducible KO ...