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Sunil Saxena

Publications and source records attributed to Sunil Saxena.

12 recordsLinked to original sources
PubMed2006

Unfolding of alanine-based peptides using electron spin resonance distance measurements.

We describe a scheme for tagging an alanine-based peptide with a Cu(II) and a nitroxide to measure unfolding transitions. The enhancement in longitudinal relaxation rate of the nitroxide due to the presence of Cu(II) was measured at physiological temperatures by pulsed electron spin resonance (ESR). The change in relaxation rate provided the average interspin distance between the Cu(II) and the nitroxide. Control experiments on a proline-based peptide verify the robustness of the method. The change in interspin distances with temperature for the alanine-based peptide is in accord with the change in helicity measured by circular dichroism. The data provide an opportunity to examine the unfolding process in polyalanine peptides. The distance in the folded state is in concordance with molecular dynamics. However, the ESR experiment measures an average distance of 17 A in the unfolded state, whereas molecular dynamics indicates a distance of 42 A if the unfolded geometry was a polyproline type II helix. Therefore, ESR demonstrates that the unfolded state of this alanine-based peptide is not an ideal extended polyproline type II helix.

Alanine↗
PubMed2006

Flexibility and lengths of bis-peptide nanostructures by electron spin resonance.

We demonstrate the use of electron spin resonance (ESR) to determine long-range distances and flexibility in water-soluble bis-peptide molecular rods. Bis-peptide oligomers with 4-8 monomer units were synthesized. ESR determined that the end-to-end length of the peptides is linearly proportional to the number of monomers. The linear shape is, therefore, easily interpreted from the data. In addition, the flexibility of the rods was quantified directly from the ESR-determined distance distribution functions. Quantitative information on chain length and flexibility is important to develop the use of these oligomers as rodlike structural elements for applications such as bivalent display of ligands and as elements of future nanoscale devices.

Electron Spin Resonance Spectroscopy↗
PubMed2005

Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells.

ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein-protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.

Amiloride↗
PubMed2005

Phosphatidylinositol 3,4,5-trisphosphate mediates aldosterone stimulation of epithelial sodium channel (ENaC) and interacts with gamma-ENaC.

Whole cell voltage clamp experiments were performed in a mouse cortical collecting duct principal cell line using patch pipettes back-filled with a solution containing phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). PIP(3) significantly increased amiloridesensitive current in control cells but not in the cells prestimulated by aldosterone. Additionally, aldosterone stimulated amiloridesensitive current in control cells, but not in the cells that expressed a PIP(3)-binding protein (Grp1-PH), which sequestered intracellular PIP(3). 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of gamma-epithelial sodium channel (gamma-ENaC) were truncated by PCRbased mutagenesis (gammaT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing alpha- and beta-ENaC only or with either gamma-ENaC or gamma(T)-ENaC. The data demonstrated that the N-terminal tail truncation significantly decreased amiloride-sensitive current and that both the N-terminal tail truncation and LY-294002 (a PI3K inhibitor) prevented ENaC translocation to the plasmamembrane. These data suggest that PIP(3) mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of gamma-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between gamma-ENaC and PIP(3).

Aldosterone↗
PubMed2005

Binding of copper(II) to thyrotropin-releasing hormone (TRH) and its analogs.

Spectroscopy (UV-Vis, (1)H NMR, ESR) and electrochemistry revealed details of the structure of the Cu(II)-TRH (pyroglutamyl-histidyl-prolyl amide) complex. The (1)H NMR spectrum of TRH has been assigned. NMR spectra of TRH in the presence of Cu(II) showed that Cu(II) initially binds TRH through the imidazole. TRH analogs, pGlu-His-Pro-OH, pGlu-(1-Me)His-Pro-amide, pGlu-His-(3,4-dehydro)Pro-amide, pGlu-His-OH, pGlu-Glu-Pro-amide, and pGlu-Phe-Pro-amide provided comparison data. The stoichiometry of the major Cu(II)-TRH complex at pH 7.45 and greater is 1:1. The conditional formation constant (in pH 9.84 borate with 12.0 mM tartrate) for the formation of the complex is above 10(5) M(-1). The coordination starts from the 1-N of the histidyl imidazole, and then proceeds along the backbone involving the deprotonated pGlu-His amide and the lactam nitrogen of the pGlu residue. The fourth equatorial donor is an oxygen donor from water. Hydroxide begins to replace the water before the pH reaches 11. Minority species with stoichiometry of Cu-(TRH)(x) (x = 2-4) probably exist at pH lower than 8.0. In non-buffered aqueous solutions, TRH acts as a monodentate ligand and forms a Cu(II)-(TRH)(4) complex through imidazole nitrogens. All the His-containing analogs behave like TRH in terms of the above properties.

Journal Article↗
PubMed2004

Steroids and exogenous gamma-ENaC subunit modulate cation channels formed by alpha-ENaC in human B lymphocytes.

Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both alpha- and beta- but not gamma-ENaC. Western blots showed that both alpha- and beta- but not gamma-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against alpha-ENaC but not by antisense oligonucleotide against beta-ENaC, indicating that only the alpha subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 microm) reduced the frequency of observance of 21-pS alpha-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from alpha- and beta-ENaC. After expression of exogenous gamma-ENaC, aldosterone again reduced the frequency of observance of the 21-pS alpha-ENaC channel but induced the appearance of a 5-pS channel, presumably a alphabetagamma-ENaC channel. In the absence of aldosterone, the alpha subunit forms an alpha-cryptic channel that is activated by stretch, and in the presence of aldosterone, beta and alpha subunits together form an active channel that is modulated by aldosterone.

Aldosterone↗
PubMed2004

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway.

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl- for gluconate(-) shifted the reversal potential, while Cl- channel blockers, 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPgammaS (a poorly hydrolyzable analog of ATP), 2',3'-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 microM suramin (a P2 receptor antagonist), and was partially blocked by 100 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca2+ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca(2+)-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

Suppression of electron spin-echo envelope modulation peaks in double quantum coherence electron spin resonance.

We show the use of the observer blind spots effect for the elimination of electron spin-echo envelope modulation (ESEEM) peaks in double quantum coherence (DQC) electron spin resonance (ESR). The suppression of ESEEM facilitates the routine and unambiguous extraction of distances from DQC-ESR spectra. This is also the first demonstration of this challenging methodology on commercial instrumentation.

Electron Spin Resonance Spectroscopy↗
PubMed2003

Amplification of xenon NMR and MRI by remote detection.

A technique is proposed in which an NMR spectrum or MRI is encoded and stored as spin polarization and is then moved to a different physical location to be detected. Remote detection allows the separate optimization of the encoding and detection steps, permitting the independent choice of experimental conditions and excitation and detection methodologies. In the initial experimental demonstration of this technique, we show that taking dilute 129Xe from a porous sample placed inside a large encoding coil and concentrating it into a smaller detection coil can amplify NMR signal. In general, the study of NMR active molecules at low concentration that have low physical filling factor is facilitated by remote detection. In the second experimental demonstration, MRI information encoded in a very low-field magnet (4-7 mT) is transferred to a high-field magnet (4.2 T) to be detected under optimized conditions. Furthermore, remote detection allows the utilization of ultrasensitive optical or superconducting quantum interference device detection techniques, which broadens the horizon of NMR experimentation.

Lasers↗
PubMed2002

Anionic phospholipids regulate native and expressed epithelial sodium channel (ENaC).

Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP(2) with anti-PIP(2) antibody, hydrolysis of PIP(2) with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP(2) and PIP(3) significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat alpha-, beta-, and gamma-ENaC. However, PIP(2) and PIP(3) did not affect surface expression of ENaC, indicating that PIP(2) and PIP(3) regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.

Adenosine Triphosphate↗
PubMed2002

Laser-polarized (129)Xe NMR and MRI at ultralow magnetic fields.

Laser-polarized (129)Xe and a high-T(c)superconducting quantum interference device (SQUID) are used to obtain magnetic resonance images in porous materials at a magnetic field of 2.3 mT, corresponding to a Larmor frequency of 27 kHz. Image resolution of 1 mm is obtained with gradients of only 1 mT/m. The resolution of xenon chemical shifts in different physicochemical environments at ultralow fields is also demonstrated. Details of the circulating flow optical pumping apparatus and the SQUID spectrometer are presented.

Journal Article↗