Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function

Abstract

The Na-K-Cl cotransporter (NKCC2) is the major salt transport pathway in the thick ascending limb of Henle's loop and is part of the molecular mechanism for blood pressure regulation. Recent screening of ∼3,000 members of the Framingham Heart Study identified nine rare independent mutations in the gene encoding NKCC2 (SLC12A1) associated with clinically reduced blood pressure and protection from hypertension (Ji WZ, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State M, Levy D, Lifton RP. Nat Genet 40: 592-599, 2008). To investigate their functional consequences, we introduced the nine mutations in human NKCC2A and examined protein function, expression, localization, regulation, and ion transport kinetics using heterologous expression in Xenopus laevis oocytes and HEK-293 cells. When expressed in oocytes, four of the mutants (T235M, R302W, L505V, and P569H) exhibited reduced transport function compared with wild-type. In HEK-293 cells, the same four mutants exhibited reduced function, and in addition N399S and P1083A had significantly lower activity than wild-type. The two most functionally impaired mutants (R302W and L505V) exhibited dramatically diminished production of complex-glycosylated protein and a decrease in or absence of plasma membrane localization, indicative of a processing defect. All of the functional human (h) NKCC2A variants were regulated by changes in oocyte volume and intracellular chloride in HEK cells, but P254A and N399S exhibited a lower constitutive activity in HEK cells. The P569H mutant exhibited a 50% reduction in sodium affinity compared with wild-type, predicting lower transport activity at lower intratubular salt concentrations, while the P254A mutant exhibited a 35% increase in rubidium affinity. We conclude that defects in NKCC2 processing, transport turnover rate, regulation, and ion affinity contribute to impaired transport function in six of the nine identified mutants, providing support for the predictive approach of Ji et al. to identify functionally important residues by sequence conservation. Such mutations in hNKCC2A are likely to reduce renal salt reabsorption, providing a mechanism for lower blood pressure.

OS9 Protein Interacts with Na-K-2Cl Co-transporter (NKCC2) and Targets Its Immature Form for the Endoplasmic Reticulum-associated Degradation Pathway

“…One emerging and promising strategy for the possible treatment of ERAD diseases is to overcome ER retention (61,62,64) because some mutant proteins such as the cystic fibrosis ⌬F508-CFTR mutant are actually functional if they exit the ER and reach their normal cellular location (63,65). In regard to NKCC2, previous reports demonstrated that, when expressed in a mammalian cell background, several NKCC2 mutants are retained in the ER (10,25,66). Moreover, although distinct cellular mechanisms may account for NKCC2 loss of function in BS1 disease, our preliminary data in the laboratory revealed that ER-associated protein degradation is the most common mechanism underpinning BS1.…”

Loop Diuretic and Ion-binding Residues Revealed by Scanning Mutagenesis of Transmembrane Helix 3 (TM3) of Na-K-Cl Cotransporter (NKCC1)

“…86 Rb ϩ Influx Assays-NKCC function was assessed by measuring 86 Rb ϩ influx into HEK cells in a 96-well plate as described previously (3,10,22), using an automated 96-well assay flux machine. To optimize cell surface expression (6), cells were grown to confluence in 96-well polylysine-coated plates and moved to a 25°C incubator 24 h prior to the experiment.…”

“…To optimize cell surface expression (6), cells were grown to confluence in 96-well polylysine-coated plates and moved to a 25°C incubator 24 h prior to the experiment. To activate NKCC1, cells were exposed to a low chloride hypotonic medium for 45 min (3,22); in the experiments of Fig. 3 Results are presented as mean Ϯ S.E.…”

Hsp70 and Hsp90 Multichaperone Complexes Sequentially Regulate Thiazide-sensitive Cotransporter Endoplasmic Reticulum-associated Degradation and Biogenesis

“…For example, reduced temperature rescues both wild type and mutant forms of immature CFTR, resulting in increased steady state CFTR abundance, enhanced ER export, and delivery to the cell surface (35). A recent study found that incubation of cells at 25°C also increased wild type NKCC2 protein abundance, indicating that, like CFTR, members of the cation chloride cotransporter family are processed in a temperature-sensitive manner (35,36). Current evidence suggests that altered client-chaperone interactions are partly responsible for the beneficial effect of low temperature on protein biogenesis (37).…”