Background:The Na-K-Cl cotransporter (NKCC1) regulates [Cl Ϫ ] in many cells.

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.

Background: Na-K-Cl cotransporters (NKCCs) are responsible for volume and chloride homeostasis and chloride transport and are targets of loop diuretic drugs. Results: Cross-links between transmembrane domains (TMs) 10 and 11/12 of NKCC1 are identified as inhibitory and stimulatory. Conclusion: Activation of NKCC1 involves movement of TM12 relative to TM10. Significance: This identifies movement of TM12 as a key step in the molecular mechanism of NKCC activation.

Episodic acidification resulting in increased acidity and inorganic aluminum (Al i ) is known to impact anadromous salmonids and has been identified as a possible cause of Atlantic salmon population decline. Sensitive life-stages such as smolts may be particularly vulnerable to impacts of short-term (days-week) acid/Al exposure, however the extent and mechanism(s) of this remain unknown. To determine if Atlantic salmon smolts are more sensitive than parr to short-term acid/Al, parr and smolts held in the same experimental tanks were exposed to control (pH 6.3-6.6, 11-37 g l −1 Al i ) and acid/Al (pH 5.0-5.4, 43-68 g l −1 Al i ) conditions in the lab, and impacts on ion regulation, stress response and gill Al accumulation were examined after 2 and 6 days. Parr and smolts were also held in cages for 2 and 6 days in a reference (Rock River, RR) and an acid/Al-impacted tributary (Ball Mountain Brook, BMB) of the West River in Southern Vermont. In the lab, losses in plasma Cl − levels occurred in both control parr and smolts as compared to fish sampled prior to the start of the study, however smolts exposed to acid/Al experienced additional losses in plasma Cl − levels (9-14 mM) after 2 and 6 days, and increases in plasma cortisol (4.3-fold) and glucose (2.9-fold) levels after 6 days, whereas these parameters were not significantly affected by acid/Al in parr. Gill Na + ,K + -ATPase (NKA) activity was not affected by acid/Al in either life-stage. Both parr and smolts held at BMB (but not RR) exhibited declines in plasma Cl − , and increases in plasma cortisol and glucose levels; these differences were significantly greater in smolts after 2 days but similar in parr and smolts after 6 days. Gill NKA activity was reduced 45-54% in both life-stages held at BMB for 6 days compared to reference fish at RR. In both studies, exposure to acid/Al resulted in gill Al accumulation in parr and smolts, with parr exhibiting two-fold greater gill Al than smolts after 6 days. Our results indicate that smolts are more sensitive than parr to short-term acid/Al. Increased sensitivity of smolts appears to be independent of a reduction in gill NKA activity and greater gill Al accumulation. Instead, increased sensitivity of smolts is likely a result of both the acquisition of seawater tolerance while still in freshwater and heightened stress responsiveness in preparation for seawater entry and residence.

SUMMARYEuryhaline teleosts such as Atlantic killifish (Fundulus heteroclitus) are able to acclimate to changing environmental salinity by tightly regulating NaCl absorption and secretion across their gills. Many studies have examined the mechanisms responsible for long-term (days) salinity acclimation; however, much remains unknown about the mechanisms of acute (hours) salinity acclimation. In this study, we tested the hypotheses that phosphorylation of the Na + -K + -Cl -cotransporter (NKCC1) located in the basolateral membrane of the gill plays a role in acute salinity acclimation and that changes in NKCC1 phosphorylation are mediated by a cAMP-protein kinase A (cAMP-PKA) pathway. Using a phospho-specific antibody, we determined the time course of changes in total and phosphorylated NKCC1 protein during acclimation to water of various salinities. Long-term (≥14 days) acclimation of killifish to seawater (SW) and 2ϫ SW resulted in 4-to 6-fold and 5-to 8-fold increases, respectively, in total gill NKCC1 protein relative to fish maintained in freshwater (FW). NKCC1 was found to be between 20% and 70% activated in fish, with lower average activation in fish acclimated to SW and 2ϫ SW compared with FW fish. Increases and decreases in the fractional level of NKCC1 phosphorylation were seen within 1 h of transfer of fish to water of higher and lower salinity, respectively, consistent with a regulatory role of phosphorylation prior to an increase in the biosynthesis of NKCC1; large changes in protein expression of NKCC1 were observed over periods of hours to days. We found that NKCC1 phosphorylation is acutely regulated in the killifish gill in response to changing environmental salinity and that phosphorylation in excised gills increases in response to forskolin stimulation of the cAMP-PKA pathway. The role of phosphorylation is further underscored by the observation that mRNA expression of sterile 20 (Ste20)-related proline-alanine-rich kinase (SPAK) changes with salinity acclimation, being 2.7-fold greater in SW-acclimated killifish relative to FW fish. Overall, these results demonstrate an important role of NKCC1 phosphorylation in the gill of Atlantic killifish during acute salinity acclimation.