Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na(+)/H(+) exchanger.

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H(+) in exchange for one extracellular Na(+). The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278-291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII.

The C-terminal tail of the plant endosomal-type NHXs plays a key role in its function and stability.

Typically, Na+/H+ antiporters (NHXs) possess a conserved N-terminus for cation binding and exchange and a hydrophilic C-terminus for regulating the antiporter activity. Plant endosomal-type NHXs play important roles in protein trafficking, as well as K+ and vesicle pH homeostasis, however the role of the C-terminal tail remains unclear. Here, the function of MnNHX6, an endosomal-type NHX in mulberry, was investigated using heterologous expression in yeast. Functional and localization analyses of C-terminal truncation and mutations in MnNHX6 revealed that the C-terminal conserved region was responsible for the function and stability of the protein and its hydrophobicity, which is a key domain requirement. Nuclear magnetic resonance spectroscopy provided direct structural evidence and yeast two-hybrid screening indicated that this functional domain was also necessary for interaction with sorting nexin 1. Our findings demonstrate that although the C-terminal tail of MnNHX6 is intrinsically disordered, the C-terminal conserved region may be an important part of the external mouth of this transporter, which controls protein function and stability by serving as an inter-molecular cork with a chain mechanism. These findings improve our understanding of the roles of the C-terminal tail of endosomal-type NHXs in plants and the ion transport mechanism of NHX-like antiporters.

Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells.

Genes encoding ion transporters that regulate ion homeostasis in soybean have not been carefully investigated. Using degenerate primers, we cloned a putative chloride channel gene (GmCLC1) and a putative Na+/H+ antiporter gene (GmNHX1) from soybean. Confocal microscopic studies using yellow fluorescent fusion proteins revealed that GmCLC1 and GmNHX1 were both localized on tonoplast. The expressions of GmCLC1 and GmNHX1 were both induced by NaCl or dehydration stress imposed by polyethylene glycol (PEG). Using mitochondrial integrity and cell death as the damage indicators, a clear alleviation under NaCl stress (but not PEG stress) was observed in both GmCLC1 and GmNHX1 transgenic cells. Using fluorescent dye staining and quenching, respectively, a higher concentration of chloride ion (Cl-) or sodium ion (Na+) was observed in isolated vacuoles in the cells of GmCLC1 and of GmNHX1 transgenic lines. Our result suggested that these vacuolar-located ion transporters function to sequester ions from cytoplasm into vacuole to reduce its toxic effects.

Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice.

Multiple Na(+)/H(+) exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na(+)/H(+) exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1(-/-)). Salivation in response to pilocarpine stimulation was reduced significantly in both Nhe1(-/-) and Nhe2(-/-) mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na(+)/K(+)/2Cl(-) cotransporter mRNA increased dramatically in Nhe1(-/-) parotid glands but not in those of Nhe2(-/-) or Nhe3(-/-) mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2(-/-) or Nhe3(-/-) mice were comparable with those of wild-type mice. In contrast, Nhe1(-/-) mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na(+) absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na(+)/K(+)/2Cl(-) cotransporter activity.

Profiling of drugs for membrane activity using liposomes as an in vitro model system.

The increasing size of chemical libraries being analyzed by high-throughput screening results in a growing number of active compounds that need to be assessed before moving forward in the drug development process. As a consequence, more rapid and highly sensitive strategies are required to accelerate the process of drug discovery without increasing the cost. Due to the fact that significant numbers of compounds from combinatorial libraries are hydrophobic in nature, approaches are needed to evaluate the potentialfor these compounds to interfere with the functions of biological membranes. The liposome system was used to detect agents that act as follows: (i) ionophores able to induce specific ion permeability, e.g., valinomycin for K+ and protonophoric uncouplers for H+; (ii) ion antiporters which exchange H+ for other ions, e.g., nigericin; (iii) agents that form low specificity ion channels in the membrane, e.g., gramicidin; and (iv) detergents and other membrane-disrupting agents. We propose using this liposome assay during the drug development process to identify compounds that have membrane activity and, as a consequence, produce a biological effect by altering the physico-chemical properties of the cell membrane rather than interacting with a protein target. Screening of a representative set of biologically-active compounds (198) indicated that the majority of systemic antimicrobial drugs, but not topical drugs, lack membrane activity in this model system.

The arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes.

In saline environments, plants accumulate Na(+) in vacuoles through the activity of tonoplast Na(+)/H(+) antiporters. The first gene for a putative plant vacuolar Na(+)/H(+) antiporter, AtNHX1, was isolated from Arabidopsis and shown to increase plant tolerance to NaCl. However, AtNHX1 mRNA was up-regulated by Na(+) or K(+) salts in plants and substituted for the homologous protein of yeast to restore tolerance to several toxic cations. To study the ion selectivity of the AtNHX1 protein, we have purified a histidine-tagged version of the protein from yeast microsomes by Ni(2+) affinity chromatography, reconstituted the protein into lipid vesicles, and measured cation-dependent H(+) exchange with the fluorescent pH indicator pyranine. The protein catalyzed Na(+) and K(+) transport with similar affinity in the presence of a pH gradient. Li(+) and Cs(+) ions were also transported with lower affinity. Ion exchange by AtNHX1 was inhibited 70% by the amiloride analog ethylisopropyl-amiloride. Our data indicate a role for intracellular antiporters in organelle pH control and osmoregulation.

What can transgenic and gene-targeted mouse models teach us about salivary gland physiology?

Thousands of genetically modified mice have been developed since the first reports of stable expression of recombinant DNA in this species nearly 20 years ago. This mammalian model system has revolutionized the study of whole-animal, organ, and cell physiology. Transgenic and gene-targeted mice have been widely used to characterize salivary-gland-specific expression and to identify genes associated with tumorigenesis. Moreover, several of these mouse lines have proved to be useful models of salivary gland disease related to impaired immunology, i.e., Sjögren's syndrome, and disease states associated with pathogens. Despite the availability of genetically modified mice, few investigators have taken advantage of this resource to better their understanding of salivary gland function as it relates to the production of saliva. In this article, we describe the methods used to generate transgenic and gene-targeted mice and provide an overview of the advantages of and potential difficulties with these models. Finally, using these mouse models, we discuss the advances made in our understanding of the salivary gland secretion process.