Secretory Carrier Membrane Protein 2 Regulates Exocytic Insertion of NKCC2 into the Cell Membrane

Abstract

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.NKCC2 is an Na-K-2Cl co-transporter protein expressed exclusively in the mammalian kidney, where it provides the major route for sodium/chloride transport across the apical plasma membrane of the thick ascending limb (1). NKCC2 is therefore a pivotal protein in renal function, and exquisitely tight regulation of the apical co-transporter activity is paramount for maintaining extracellular fluid volume and acid-base homeostasis (2). It is the target of loop-diuretics extensively used in the treatment of edematous states and hypertension (3). Furthermore, recent work implicates NKCC2 in the dysfunction of blood pressure regulation, raising new hypotheses regarding the underlying mechanisms behind some types of essential hypertension (4 -7). Most importantly, inactivating mutations of the NKCC2 gene in humans causes Bartter syndrome type 1, a life-threatening kidney disease (8). However, despite this importance, little is known about NKCC2 regulation in renal cells, mainly because of the difficul...

Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2

“…A Myc epitope-tagged wild-type or mutant variants of NKCC2 were transiently transfected into OKP cells and HEK cells, and the degree of protein processing was determined by immunoblot analysis of total proteins. In agreement with our previously published data (20,23,24), when wild-type NKCC2 is exogenously expressed in renal cultured cells, both the core (immature) and complex glycosylated (mature) forms are observed (Fig. 1, B and C), the latter being the more abundant (20,23,24 (Fig.…”

“…In agreement with our previously published data (20,23,24), when wild-type NKCC2 is exogenously expressed in renal cultured cells, both the core (immature) and complex glycosylated (mature) forms are observed (Fig. 1, B and C), the latter being the more abundant (20,23,24 (Fig. 1B, lanes 3-5), indicating that these motifs do not play a crucial role in NKCC2 maturation.…”

“…20 mM NH 4 Cl was then added to the medium, inducing a very rapid initial cellular alkalinization followed by a pH i recovery. The initial rate of intracellular pH recovery (dpH i /dt) was measured over the first 20 s of records, as reported earlier (20,23,24). The ⌬pH i caused by NH 4 Cl addition was used to calculate the cell buffer capacity, which was not different between the studied groups (data not shown).…”

“…On the basis of these data, we concluded that the two di-leucine-like motifs, 1038 mutations on the NKCC2 surface level, we sought to determine whether this was associated with changes in NKCC2 co-transport activity. To address this, Na-K ϩ (NH 4 ϩ )-2Cl transport activity was assessed by estimating the rate of intracellular acidification caused by entry into the cells of NH 4 ϩ via this transport mechanism after abrupt application of NH 4 Cl to the cells (20,23,24 (Fig. 2C).…”

“…Calibration of the 2Ј,7Ј-bis (carboxyethyl)-5,6-carboxyfluorescein excitation ratio was accomplished using the nigericin technique. Forty-eight hours posttransfection, Na-K-2Cl cotransport activity was measured as bumetanide-sensitive NH 4 influx, as previously described (20,23,24). Cells were first bathed at 37°C in a CO 2 -free Hepes/ Tris-buffered medium containing 135 mM NaCl, 10 mM Hepes (pH 7.4), 5 mM KCl, 1 mM MgCl 2 , 0.8 mM KH 2 PO 4 , 0.2 mM K 2 HOP 4 , 1 mM CaCl 2 , and 10 mM BaCl 2 to measure base-line pH i .…”