PubMed Health⌕ Search

Biomedical subjects

Rajini Rao

Publications and source records attributed to Rajini Rao.

15 recordsLinked to original sources
PubMed2006

Vestibular hair bundles control pH with (Na+, K+)/H+ exchangers NHE6 and NHE9.

In hair cells of the inner ear, robust Ca2+/H+ exchange mediated by plasma-membrane Ca2+-ATPase would rapidly acidify mechanically sensitive hair bundles without efficient removal of H+. We found that, whereas the basolateral membrane of vestibular hair cells from the frog saccule extrudes H+ via an Na+-dependent mechanism, bundles rapidly remove H+ in the absence of Na+ and HCO3(-), even when the soma is acidified. K+ was fully effective and sufficient for H+ removal; in contrast, Rb+ failed to support pH recovery. Na+/H+-exchanger isoform 1 (NHE1) was present on hair-cell soma membranes and was likely responsible for Na+-dependent H+ extrusion. NHE6 and NHE9 are organellar isoforms that can appear transiently on plasma membranes and have been proposed to mediate K+/H+ exchange. We identified NHE6 in a subset of hair bundles; NHE9 was present in all bundles. Heterologous expression of these isoforms in yeast strains lacking endogenous exchangers conferred pH-dependent tolerance to high levels of KCl and NaCl. NHE9 preferred cations in the order K+, Na+ >> Rb+, consistent with the relative efficacies of these ions in promoting pH recovery in hair bundles. Electroneutral K+/H+ exchange, which we propose is performed by NHE9 in hair bundles, exploits the high-K+ endolymph, responds only to pH imbalance across the bundle membrane, is unaffected by the +80 mV endocochlear potential, and uses mechanisms already present in the ear for K+ recycling. This mechanism allows the hair cell to remove H+ generated by Ca2+ pumping without ATP hydrolysis in the cell.

Amino Acid Sequence↗
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

A novel isoform of the secretory pathway Ca2+,Mn(2+)-ATPase, hSPCA2, has unusual properties and is expressed in the brain.

Unlike lower eukaryotes, mammalian genomes have a second gene, ATP2C2, encoding a putative member of the family of secretory pathway Ca2+,Mn(2+)-ATPases, SPCA2. Human SPCA2 shares 64% amino acid identity with the protein defective in Hailey Hailey disease, hSPCA1. We show that human SPCA2 (hSPCA2) has a more limited tissue distribution than hSPCA1, with prominent protein expression in brain and testis. In primary neuronal cells, endogenous SPCA2 has a highly punctate distribution that overlaps with vesicles derived from the trans-Golgi network and is thus different from the compact perinuclear distribution of hSPCA1 seen in keratinocytes and nonpolarized cells. Heterologous expression in a yeast strain lacking endogenous Ca2+ pumps reveals further functional differences from hSPCA1. Although the Mn(2+)-specific phenotype of hSPCA2 is similar to that of hSPCA1, Ca2+ ions are transported with much poorer affinity, resulting in only weak complementation of Ca(2+)-specific yeast phenotypes. These observations suggest that SPCA2 may have a more specialized role in mammalian cells, possibly in cellular detoxification of Mn2+ ions, similar to that in yeast. We point to the close links between manganese neurotoxicity and Parkinsonism that would predict an important physiological role for SPCA2 in the brain.

Amino Acid Sequence↗
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↗
PubMed2004

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases.

The baker's yeast Saccharomyces cerevisiae is a well-developed, versatile, and widely used model organism. It offers a compact and fully sequenced genome, tractable genetics, simple and inexpensive culturing conditions, and, importantly, a conservation of basic cellular machinery and signal transducing pathways with higher eukaryotes. In this review, we describe recent technical advances in the heterologous expression of proteins in yeast and illustrate their application to the study of the Ca(2+) homeostasis machinery, with particular emphasis on Ca(2+)-transporting ATPases. Putative Ca(2+)-ATPases in the newly sequenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of an engineered yeast strain lacking endogenous Ca(2+) pumps. High-throughput screens of mutant phenotypes to identify side chains critical for ion transport and selectivity have facilitated structure-function analysis, and genomewide approaches may be used to dissect cellular pathways involved in Ca(2+) transport and trafficking. The utility of the yeast system is demonstrated by rapid advances in the study of the emerging family of Golgi/secretory pathway Ca(2+),Mn(2+)-ATPases (SPCA). Functional expression of human SPCA1 in yeast has provided insight into the physiology, novel biochemical characteristics, and subcellular localization of this pump. Haploinsufficiency of SPCA1 leads to Hailey-Hailey disease (HDD), a debilitating blistering disorder of the skin. Missense mutations, identified in patients with HHD, may be conveniently assessed in yeast for loss-of-function phenotypes associated with the disease.

Animals↗
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↗
PubMed2003

Packing interactions between transmembrane helices alter ion selectivity of the yeast Golgi Ca2+/Mn2+-ATPase PMR1.

PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.

Amino Acid Sequence↗
PubMed2003

Antifungal activity of amiodarone is mediated by disruption of calcium homeostasis.

The antiarrhythmic drug amiodarone was recently demonstrated to have novel broad range fungicidal activity. We provide evidence that amiodarone toxicity is mediated by disruption of Ca2+ homeostasis in Saccharomyces cerevisiae. In mutants lacking calcineurin and various Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels (Cch1/Mid1 and Yvc1), and exchangers (Vcx1), amiodarone sensitivity correlates with cytoplasmic calcium overload. Measurements of cytosolic Ca2+ by aequorin luminescence demonstrate a biphasic response to amiodarone. An immediate and extensive calcium influx was observed that was dose-dependent and correlated with drug sensitivity. The second phase consisted of a sustained release of calcium from the vacuole via the calcium channel Yvc1 and was independent of extracellular Ca2+ entry. To uncover additional cellular pathways involved in amiodarone sensitivity, we conducted a genome-wide screen of nearly 5000 single-gene yeast deletion mutants. 36 yeast strains with amiodarone hypersensitivity were identified, including mutants in transporters (pmr1, pdr5, and vacuolar H+-ATPase), ergosterol biosynthesis (erg3, erg6, and erg24), intracellular trafficking (vps45 and rcy1), and signaling (ypk1 and ptc1). Of three mutants examined (vps45, vma3, and rcy1), all were found to have defective calcium homeostasis, supporting a correlation with amiodarone hypersensitivity. We show that low doses of amiodarone and an azole (miconazole, fluconazole) are strongly synergistic and exhibit potent fungicidal effects in combination. Our findings point to the potentially effective application of amiodarone as a novel antimycotic, particularly in combination with conventional antifungals.

Amiodarone↗
PubMed2002

Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis.

The internal environment of the ER is regulated to accommodate essential cellular processes, yet our understanding of this regulation remains incomplete. Cod1p/Spf1p belongs to the widely conserved, uncharacterized type V branch of P-type ATPases, a large family of ion pumps. Our previous work suggested Cod1p may function in the ER. Consistent with this hypothesis, we localized Cod1p to the ER membrane. The cod1Delta mutant disrupted cellular calcium homeostasis, causing increased transcription of calcium-regulated genes and a synergistic increase in cellular calcium when paired with disruption of the Golgi apparatus-localized Ca2+ pump Pmr1p. Deletion of COD1 also impaired ER function, causing constitutive activation of the unfolded protein response, hypersensitivity to the glycosylation inhibitor tunicamycin, and synthetic lethality with deletion of the unfolded protein response regulator HAC1. Expression of the Drosophila melanogaster homologue of Cod1p complemented the cod1Delta mutant. Finally, we demonstrated the ATPase activity of the purified protein. This study provides the first biochemical characterization of a type V P-type ATPase, implicates Cod1p in ER function and ion homeostasis, and indicates that these functions are conserved among Cod1p's metazoan homologues.

ATP-Binding Cassette Transporters↗
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↗
PubMed2001

Functional expression in yeast of the human secretory pathway Ca(2+), Mn(2+)-ATPase defective in Hailey-Hailey disease.

The discovery and biochemical characterization of the secretory pathway Ca(2+)-ATPase, PMR1, in Saccharomyces cerevisiae, has paved the way for identification of PMR1 homologues in many species including rat, Caenorhabditis elegans, and Homo sapiens. In yeast, PMR1 has been shown to function as a high affinity Ca(2+)/Mn(2+) pump and has been localized to the Golgi compartment where it is important for protein sorting, processing, and glycosylation. However, little is known about PMR1 homologues in higher organisms. Loss of one functional allele of the human gene, hSPCA1, has been linked to Hailey-Hailey disease, characterized by skin ulceration and improper keratinocyte adhesion. We demonstrate that expression of hSPCA1 in yeast fully complements pmr1 phenotypes of hypersensitivity to Ca(2+) chelators and Mn(2+) toxicity. Similar to PMR1, epitope-tagged hSPCA1 also resides in the Golgi when expressed in yeast or in chinese hamster ovary cells. (45)Ca(2+) transport by hSPCA1 into isolated yeast Golgi vesicles shows an apparent Ca(2+) affinity of 0.26 microm, is inhibitable by Mn(2+), but is thapsigargin-insensitive. In contrast, heterologous expression of vertebrate sarcoplasmic reticulum and plasma membrane Ca(2+)-ATPases in yeast complement the Ca(2+)- but not Mn(2+)-related phenotypes of the pmr1-null strain, suggesting that high affinity Mn(2+) transport is a unique feature of the secretory pathway Ca(2+)-ATPases.

Animals↗
PubMed1999

The yeast endosomal Na+/H+ exchanger, Nhx1, confers osmotolerance following acute hypertonic shock.

Osmotolerance in yeast is regulated by at least two distinct mechanisms. The acquired response occurs following long-term exposure to hypertonic medium and requires the induction of the HOG-MAP (high-osmolarity glycerol mitogen-activated protein) kinase cascade to increase levels of the osmolyte glycerol. The acute response occurs following sudden exposure to high osmotica and appears to be dependent on normal vacuole function. In this study it is reported that the yeast endosomal/prevacuolar Na+/H+ exchanger Nhx1 contributes to osmotolerance following sudden exposure to hyperosmotic media. Vacuolar shrinkage and recovery in response to osmotic shock was altered in the (delta)nhx1 null mutant. Our results also show that the osmotolerance conferred by Nhx1 contributes to the postdiauxic/stationary-phase resistance to osmotic stress and allows for the continued growth of cells until the acquired osmotolerance response can occur.

Cation Transport Proteins↗