Biomedical subjects

Christopher L Brett

Publications and source records attributed to Christopher L Brett.

8 recordsLinked to original sources

PubMed2006

Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking.

Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.

Amino Acid Sequence↗

PubMed2006

Does the proteome encode organellar pH?

Inherent to the proteome itself, may be information that enables proteins to buffer pH at a level that promotes their own function within a specialized compartment. We observe that the distribution of computed isoelectric points in the yeast proteome matches experimentally derived organellar pH estimates across distinct subcellular compartments. This raises an interesting evolutionary question: did the pI of proteins and the pH of organelles co-evolve to optimize function?

Animals↗

PubMed2005

NHERF family and NHE3 regulation.

The intestinal and renal proximal tubule brush border (BB) Na+-H+ exchanger NHE3 binds to members of the NHERF (Na+-H+ exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable series of proteins in NHE3-containing complexes in a dynamic manner that is altered by changes in signal transduction which affects NHE3 activity. The specific roles of these proteins in terms of NHE3 regulation as well as interactions with each other and with their many other substrates are only now being defined. Specificity for only one member of the NHERF family in one example of NHE3 regulation, inhibition by elevation in cGMP, is used to describe how NHERF family proteins are involved in NHE3 complex formation and its regulation. In this case, NHERF2 directly binds cGKII in the brush border to form an NHE3 complex, with cGKII also associating with the BB via its myristoylation.

Animals↗

PubMed2005

The yeast endosomal Na+K+/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking.

The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway. However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified. Herein we describe the use of novel, compartment-specific pH indicators to demonstrate that yeast Nhx1, an endosomal member of the ubiquitous NHE family of Na+/H+ exchangers, regulates luminal and cytoplasmic pH to control vesicle trafficking out of the endosome. Loss of Nhx1 confers growth sensitivity to low pH stress, and concomitant acidification and trafficking defects, which can be alleviated by weak bases. Conversely, weak acids cause wild-type yeast to present nhx1Delta trafficking phenotypes. Finally, we report that Nhx1 transports K+ in addition to Na+, suggesting that a single mechanism may responsible for both pH and K+-dependent endosomal processes. This presents the newly defined family of eukaryotic endosomal NHE as novel targets for pharmacological inhibition to alleviate pathological states associated with organellar alkalinization.

Biological Transport↗

PubMed2005

Evolutionary origins of eukaryotic sodium/proton exchangers.

More than 200 genes annotated as Na+/H+ hydrogen exchangers (NHEs) currently reside in bioinformation databases such as GenBank and Pfam. We performed detailed phylogenetic analyses of these NHEs in an effort to better understand their specific functions and physiological roles. This analysis initially required examining the entire monovalent cation proton antiporter (CPA) superfamily that includes the CPA1, CPA2, and NaT-DC families of transporters, each of which has a unique set of bacterial ancestors. We have concluded that there are nine human NHE (or SLC9A) paralogs as well as two previously unknown human CPA2 genes, which we have named HsNHA1 and HsNHA2. The eukaryotic NHE family is composed of five phylogenetically distinct clades that differ in subcellular location, drug sensitivity, cation selectivity, and sequence length. The major subgroups are plasma membrane (recycling and resident) and intracellular (endosomal/TGN, NHE8-like, and plant vacuolar). HsNHE1, the first cloned eukaryotic NHE gene, belongs to the resident plasma membrane clade. The latter is the most recent to emerge, being found exclusively in vertebrates. In contrast, the intracellular clades are ubiquitously distributed and are likely precursors to the plasma membrane NHE. Yeast endosomal ScNHX1 was the first intracellular NHE to be described and is closely related to HsNHE6, HsNHE7, and HsNHE9 in humans. Our results link the appearance of NHE on the plasma membrane of animal cells to the use of the Na+/K(+)-ATPase to generate the membrane potential. These novel observations have allowed us to use comparative biology to predict physiological roles for the nine human NHE paralogs and to propose appropriate model organisms in which to study the unique properties of each NHE subclass.

Amino Acid Sequence↗

PubMed2003

Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast.

Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.

Base Sequence↗

PubMed2002

Regulation of Cl--HCO3- exchangers by cAMP-dependent protein kinase in adult rat hippocampal CA1 neurons.

The contributions of HCO(3)(-)-dependent, DIDS-sensitive mechanisms to the maintenance of steady-state pH(i), and the regulation of their activities by cAMP-dependent protein kinase (PKA), were investigated in CA1 neurons with the H(+)-sensitive fluorophore, BCECF. The addition of HCO(3)(-)/CO(2) to neurons with "low" (pH(i) < or = 7.20) and "high" (pH(i) > 7.20) initial pH(i) values under Hepes-buffered conditions, increased and decreased steady-state pH(i), respectively. Conversely, under HCO(3)(-)/CO(2)-buffered conditions, DIDS caused pH(i) to decrease and increase in neurons with low and high initial pH(i) values, respectively. In the presence, but not the absence, of HCO(3)(-), the PKA inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS; 50 microM) evoked DIDS-sensitive increases and decreases in pH(i) in neurons with low and high initial pH(i) values, respectively. In contrast, in neurons with low initial pH(i) values, activation of PKA with the Sp isomer of cAMPS (Sp-cAMPS; 25 microM) elicited increases in pH(i) that were smaller in the presence than in the absence of HCO(3)(-), whereas in neurons with high initial pH(i) values, Sp-cAMPS-evoked rises in pH(i) were larger in the presence than in the absence of HCO(3)(-); the differences between the effects of Sp-cAMPS on pH(i) under the different buffering conditions were attenuated by DIDS. Consistent with the possibility that changes in the activities of HCO(3)(-)-dependent, DIDS-sensitive mechanisms contribute to the steady-state pH(i) changes evoked by the PKA modulators, in neurons with initial pH(i) values < or = 7.20, Rp-cAMPS concurrently inhibited Na(+)-independent Cl(-)-HCO(3)(-) exchange and stimulated Na(+)-dependent Cl(-)-HCO(3)(-) exchange; in contrast, Sp-cAMPS concurrently stimulated Na(+)-independent Cl(-)-HCO(3)(-) exchange and inhibited Na(+)-dependent Cl(-)-HCO(3)(-) exchange. Data from a limited number of neurons with initial pH(i) values > 7.20 suggested that the directions of the reciprocal changes in anion exchange activities (inhibition or stimulation) evoked by Rp- and Sp-cAMPS may be opposite in cells with low vs. high resting pH(i) values. Taken together, the results indicate that the effects of modulating PKA activity on steady-state pH(i) in rat CA1 neurons under HCO(3)(-)/CO(2)-buffered conditions reflect not only changes in Na(+)-H(+) exchange activity but also changes in Na(+)-dependent and Na(+)-independent Cl(-)-HCO(3)(-) exchange activity that, in turn, may be dependent upon the initial pH(i).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2002

Human Na(+)/H(+) exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria.

Since the discovery of the first intracellular Na(+)/H(+) exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na(+) into the vacuole, regulates intravesicular pH, and contributes to vacuolar biogenesis. In contrast, the human homolog of Nhx1, Na(+)/H(+) exchanger isoform 6 (NHE6), has been reported to localize to mitochondria when transiently expressed as a fusion with green fluorescent protein. This result warrants reevaluation because it conflicts with predictions from phylogenetic analyses. Here we demonstrate that when epitope-tagged NHE6 is transiently expressed in cultured mammalian cells, it does not colocalize with mitochondrial markers. It also does not colocalize with markers of the lysosome, late endosome, trans-Golgi network, or Golgi cisternae. Rather, NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane. These results suggest that, like its homologs in yeast and plants, NHE6 is an endosomal Na(+)/H(+) exchanger that may regulate intravesicular pH and volume and contribute to lysosomal biogenesis.

Animals↗