Proposed topology diagram of NCC, an SLC12 cotransporter

Proposed topology diagram of NCC, an SLC12 cotransporter

Cellular and Molecular Physiology of SLC12 Cotransporters

The primary goal of our research is to understand how sodium, chloride, and potassium transport is coordinated in the kidney and other organs. Most of our work is centered on the renal cell biology and molecular physiology of SLC12 cation chloride cotransporters, which transport sodium and/or potassium with chloride in an electroneutral manner. Two members of the family, the bumetanide-sensitive Na-K-2Cl cotransporter NKCC2 (SLC12A1) and the thiazide-sensitive NaCl cotransporter NCC (SLC12A3), mediate salt reabsorption in the loop of Henle and distal convoluted tubule, respectively. They also appear to regulate distal potassium secretion by controlling the delivery of electrolytes to downstream nephron segments, such as the collecting duct. Thus, they play an important role in extracellular fluid volume, blood pressure, and potassium homeostasis. Loss of function mutations of NKCC2 and NCC cause Bartter and Gitelman syndromes, two hereditary salt wasting hypokalemic nephropathies. Conversely, gain of function mutations of NCC cause Familial Hyperkalemic Hypertension (FHHt), a rare chloride-dependent disorder of elevated blood pressure and potassium levels that can be cured by thiazide diuretics.

  A model of the WNK-SPAK/OSR1 pathway

A model of the WNK-SPAK/OSR1 pathway

Regulation of NCC and NKCC2 by the WNK-SPAK/OSR1 Pathway

Mutations in four genes cause FHHt. Two of these genes encode the serine-threonine kinases WNK1 and WNK4; the other two genes are KLHL3 and CUL3, which form part of an E3 ubiquitin ligase complex that regulates WNK1 and WNK4 abundance. In all cases, FHHt gene mutations increase WNK abundance and activity. The WNKs act as critical components of a signaling cascade that triggers NCC and NKCC2 phosphorylation by the effector kinases SPAK and OSR1. Phosphorylation appears to be one of the primary mechanisms by which SLC12 cotransporters are physiologically regulated. A major goal of our efforts is to identify new disease-relevant pathways that control the activity of WNK kinases and their downstream substrates.

Molecular Basis of Bartter and Gitelman Syndromes:
NCC/NKCC2 Biogenesis and ERAD

  Hsp70 and Hsp90 are molecular chaperones that regulate the early stages of NCC biogenesis

Hsp70 and Hsp90 are molecular chaperones that regulate the early stages of NCC biogenesis

Cation chloride cotransporters are polytopic membrane glycoproteins, consisting of 12 transmembrane segments flanked by two large cytosolic domains. Given their complex topology, many of the SLC12 cotransporters are prone to misfolding and disposal by endoplasmic reticulum associated degradation (ERAD). Working with the Pittsburgh Center for Kidney Research, we used mass spectrometry and yeast genetics to identify a network of molecular chaperones, co-chaperones, and E3 ubiquitin ligases that regulate NCC and NKCC2 folding and ERAD. We established that disease-associated misfolded forms of NCC that cause Gitelman syndrome interact more strongly with the Hsp70 system, targeting it for proteasomal degradation. We are currently applying a variety of new methods to further define the network that facilitates SLC12 cotransporter ER quality control and surface delivery.

 

Support

Our research is supported by the National Institutes of Health and the US Department of Veterans Affairs.