RESUMEN
The transition from freshwater to seawater represents a physiological challenge for Atlantic salmon smolts preparing for downstream migration. Stressors occurring during downstream migration to the ocean impair the ability of smolts to maintain osmotic/ionic homeostasis in seawater. The molecular mechanisms underlying this interaction are not fully understood, especially at the organ level. We combined RNA-Seq with measures of whole-animal homeostasis to examine gene expression dynamics in the gills of smolts associated with impaired seawater tolerance after an aquaculture-related stressor. Smolts were given a 24â h seawater tolerance test before and after exposure to an acute handling/confinement stress. RNA-Seq followed by differential expression and weighted gene correlation network analysis (WGCNA) was used to quantify the transcriptional response of the gill to handling/confinement stress, seawater and their interaction. Exposure to acute stress was associated with a general stress response and impaired osmotic/ionic homeostasis in seawater. We identified gene networks in the gill exhibiting response to acute stress alone, seawater alone, and others exhibiting combined effects of both stress and seawater. Our findings indicate that acute handling/confinement stress increases the intensity of seawater-related gene expression and suggest that increased investment in mechanisms related to ion transport may be part of a compensatory response to impaired seawater tolerance in smolts.
Asunto(s)
Branquias , Salmo salar , Animales , Agua Dulce , Branquias/metabolismo , Salmo salar/genética , Salmo salar/metabolismo , Agua de Mar , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu(2+) inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met(382) of TM3 are involved in a Cu(2+)-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.
Asunto(s)
Transporte Iónico , Simportadores de Cloruro de Sodio-Potasio/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Cloruros/química , Cobre/química , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Homeostasis , Humanos , Iones , Cinética , Microscopía Confocal , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fenantrolinas/química , Fosforilación , Estructura Terciaria de Proteína , Radioisótopos de Rubidio/química , Homología de Secuencia de Aminoácido , Miembro 2 de la Familia de Transportadores de Soluto 12/química , Miembro 2 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
The Na-K-Cl cotransporter (NKCC) couples the movement of Na(+), K(+), and Cl(-) ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(-) -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.
Asunto(s)
Cisteína , ADN Complementario/metabolismo , Epítopos/biosíntesis , Miembro 2 de la Familia de Transportadores de Soluto 12/biosíntesis , ADN Complementario/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Epítopos/genética , Células HEK293 , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.
Asunto(s)
Proteínas de Peces/química , Modelos Moleculares , Multimerización de Proteína , Simportadores de Cloruro de Sodio-Potasio/química , Animales , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Movimiento , Tiburones , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
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 â¼3,000 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.
Asunto(s)
Riñón/metabolismo , Potasio/metabolismo , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Sodio/metabolismo , Análisis de Varianza , Animales , Transporte Biológico/genética , Western Blotting , Células HEK293 , Humanos , Mutación , Oocitos , Miembro 1 de la Familia de Transportadores de Soluto 12 , XenopusRESUMEN
We examined the physiological, molecular, and cellular mechanisms of impaired ion regulation in Atlantic salmon, Salmo salar, smolts following acute acid and aluminum (Al) exposure. Smolts were exposed to: control (pH 6.5, 3.4 micrpg l(-1) Al), acid and low Al (LAl: pH 5.4, 11 microg l(-1) Al), acid and moderate Al (MAl: pH 5.3, 42 microg l(-1) Al), and acid and high Al (HAl: pH 5.4, 56 microg l(-1) Al) for two and six days. At each time-point, smolts were sampled directly from freshwater treatment tanks and after a 24h seawater challenge. Exposure to acid/MAl and acid/HAl led to accumulation of gill Al, substantial alterations in gill morphology, reduced gill Na(+)/K(+)-ATPase (NKA) activity, and impaired ion regulation in both freshwater and seawater. Exposure to acid/MAl for six days also led to a decrease in gill mRNA expression of the apical Cl(-) channel (cystic fibrosis transmembrane conductance regulator I), increased apoptosis upon seawater exposure, an increase in the surface expression of mitochondria-rich cells (MRCs) within the filament epithelium of the gill, but reduced abundance of gill NKA-positive MRCs. By contrast, smolts exposed to acid and the lowest Al concentration exhibited minor gill Al accumulation, slight morphological modifications in the gill, and impaired seawater tolerance in the absence of a detectable effect on freshwater ion regulation. These impacts were accompanied by decreased cell proliferation, a slight increase in the surface expression of MRCs within the filament epithelium, but no impact on gill apoptosis or total MRC abundance was observed. However, MRCs in the gills of smolts exposed to acid/LAl exhibited morphological alterations including decreased size, staining intensity, and shape factor. We demonstrate that the seawater tolerance of Atlantic salmon smolts is extremely sensitive to acute exposure to acid and low levels of Al, and that the mechanisms underlying this depend on the time-course and severity of Al exposure. We propose that when smolts are exposed to acid and moderate to high Al concentrations, impaired seawater tolerance results from extensive gill Al accumulation, damage to the epithelium, reduced MRC and transport protein abundance, and a synergistic stimulation of apoptosis in the gill upon seawater exposure. When smolts are exposed to acid and low levels of Al, loss of seawater tolerance appears to be independent of these mechanisms and may result instead from a shift in the phenotype of MRCs present in the gill epithelium.
Asunto(s)
Ácidos/toxicidad , Aluminio/toxicidad , Branquias/citología , Branquias/efectos de los fármacos , Salmo salar/fisiología , Agua de Mar , Contaminantes Químicos del Agua/toxicidad , Lluvia Ácida/toxicidad , Animales , Secuencia de Bases , Caspasa 3/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Branquias/enzimología , Branquias/metabolismo , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Mitocondrias/metabolismo , Mortalidad , Salmo salar/crecimiento & desarrollo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Euryhaline 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 (>or=14 days) acclimation of killifish to seawater (SW) and 2x 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 2x 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.
Asunto(s)
Aclimatación , Fundulidae/fisiología , Branquias/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Animales , Regulación hacia Abajo , Fundulidae/metabolismo , Fosforilación , ARN Mensajero/genética , Salinidad , Simportadores de Cloruro de Sodio-Potasio/genética , Regulación hacia ArribaRESUMEN
Episodic acidification resulting in increased acidity and inorganic aluminum (Al(i)) is known to interfere with the parr-smolt transformation of Atlantic salmon (Salmo salar), and has been implicated as a possible cause of population decline. To determine the extent and mechanism(s) by which short-term acid/Al exposure compromises smolt development, Atlantic salmon smolts were exposed to either control (pH 6.7-6.9) or acid/Al (pH 5.4-6.3, 28-64 microgl(-1) Al(i)) conditions for 2 and 5 days, and impacts on freshwater (FW) ion regulation, seawater (SW) tolerance, plasma hormone levels and stress response were examined. Gill Al concentrations were elevated in all smolts exposed to acid/Al relative to controls confirming exposure to increased Al(i). There was no effect of acid/Al on plasma ion concentrations in FW however, smolts exposed to acid/Al followed by a 24h SW challenge exhibited greater plasma Cl(-) levels than controls, indicating reduced SW tolerance. Loss of SW tolerance was accompanied by reductions in gill Na(+),K(+)-ATPase (NKA) activity and Na(+),K(+),2Cl(-) (NKCC) cotransporter protein abundance. Acid/Al exposure resulted in decreased plasma insulin-like growth factor (IGF-I) and 3,3',5'-triiodo-l-thyronine (T(3)) levels, whereas no effect of treatment was seen on plasma cortisol, growth hormone (GH), or thyroxine (T(4)) levels. Acid/Al exposure resulted in increased hematocrit and plasma glucose levels in FW, but both returned to control levels after 24h in SW. The results indicate that smolt development and SW tolerance are compromised by short-term exposure to acid/Al in the absence of detectable impacts on FW ion regulation. Loss of SW tolerance during short-term acid/Al exposure likely results from reductions in gill NKA and NKCC, possibly mediated by decreases in plasma IGF-I and T(3).
Asunto(s)
Aclimatación/efectos de los fármacos , Ácidos/farmacología , Aluminio/farmacología , Sistema Endocrino/fisiología , Muda/efectos de los fármacos , Salmo salar/fisiología , Agua de Mar , Aclimatación/fisiología , Animales , Glucemia/análisis , Glucemia/efectos de los fármacos , Cloruros/sangre , Branquias/enzimología , Branquias/metabolismo , Hormona del Crecimiento/sangre , Hidrocortisona/sangre , Factor I del Crecimiento Similar a la Insulina/análisis , Salmo salar/sangre , Salmo salar/crecimiento & desarrollo , Salmo salar/metabolismo , Sodio/sangre , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Factores de TiempoRESUMEN
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 microgl(-1) Al(i)) and acid/Al (pH 5.0-5.4, 43-68 microgl(-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.