Research Project
10280368
ATP1A1 is the only Na/K-ATPase (NKA) isoform expressed in the kidney. As stated in physiology textbooks, NKA is the enzymatic machinery that powers energy storage in the form of a transmembrane Na+ gradient, which is essential for renal salt handling and the control of blood pressure. In the renal proximal tubule (RPT), activation of NKA-mediated classic ion transport function decreases natriuresis through activation of both basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers a cellular redistribution of both NKA and NHE3 in RPT cells, which decreases Na+ uptake. Hence, RPT NKA simultaneously serves two opposing roles in Na+ handling: anti-natriuretic through its classic ATPase-driven ion transport function, and natriuretic through its more recently recognized receptor/signaling function. To date, the relative contributions of these two NKA functions to the net RPT Na+ handling in vivo is a fundamentally and therapeutically essential question that has been virtually impossible to answer. NKA signaling, which is both distinct and independent from NKA classic enzymatic ion-transporting function, was first brought to the attention of the scientific community by the work of Dr. Zijian Xie in ouabain-treated cardiac myocytes and renal epithelial cells. Mechanistically, binding of NKA specific ligands such as the cardiotonic steroid (CTS) ouabain activates Src, resulting in the activation of multiple protein/lipid kinases and the generation of intracellular second messengers. Numerous groups around the world have expanded the concept of non- enzymatic signaling function of NKA, while we have focused on the mechanism by which NKA is engaged in direct interaction with several signaling and scaffolding proteins including Src. This has allowed us to develop ATP1A1 mutants with intact enzymatic function but defective ability of interaction with signaling partners. Critically, we have developed a hypomorphic mouse (RPT?1-/-) with a RPT-specific reduction of 70% of NKA ?1. The hyper-reabsorptive renal phenotype of this mouse suggests that NKA signaling is not only physiologically relevant, but also functionally prevalent in the regulation of RPT Na+ handling. We have established feasibility of RPT-specific rescue of the hypomorphic RPT?1-/- mouse with either wild-type or Src-binding null mutant forms of NKA, and propose to use those new tools to test the central hypothesis that NKA ?1 (ATP1A1) exerts a tonic inhibition of apical NHE3 and basolateral Na+ transporters in the RPT. We further surmise that this regulatory mechanism has a prevalent regulatory role in RPT Na+ handling (Aim 1), depends on NKA ?1/Src interaction (Aim 2), and enables NKA ?1 ligands such as CTS to modulate RPT Na+ reabsorption (Aim 3). Successful completion of the proposed investigation shall reveal a hitherto unrecognized regulatory mechanism of salt handling by RPT NKA ?1, the molecular basis of this regulation, an integrated compensatory transport network, and their impact on renal physiology and the development of salt sensitivity.
