Contact

Location: 3.029V

Department

Cellular and Integrative Physiology

James Stockand, Ph.D.

Professor

Education

B.S.,Texas A & M University, 1991
Ph.D., Univ. of Texas Health Science Center at Houston, 1996

Research

Cell model of a Principal cell

Cell model of a Principal cell. Aldosterone modulates ENaC in these cells by first affecting gene expression through the mineralocorticoid receptor and inducing expression of the Ki-Ras and Sgk signaling proteins, which then signal to ENaC to modulate ion channel activity.
My laboratory uses a number of contemporary methodologies, including electrophysiology, molecular biology, biochemistry, genomics and proteomics, and fluorescence microscopy to investigate regulation of ENaC and aldosterone signaling. We routinely use yeast, bacteria, immortalized cell lines and animals in this regard. Our lab is equipped to perform real-time measurement of ion channel activity and fluorescence imaging, and performs biophysical analysis of ion channel activity and gating, creates point mutations and chimeric proteins and genes, investigates cellular signal transduction, reconstitutes signaling pathways in heterologous systems, and determines the molecular mechanisms modulating gene expression, and uses the power of yeast genetics in addition to immortalized mammalian cells to investigate ENaC targeting and regulation. The pictures below give some idea of the methods we employ.

Flourescence Microscopy

Targeting ENaC to different cellular locales. Fusion proteins were created by tagging the cytosolic domains of ENaC with GFP. Cellular localization was determined in a heterologous system by overexpressing these chimeric fusion proteins.
Electrophysiology

Single channel current recordings for ENaC in cell attached patches. A. ENaC activity in the presence of aldosterone is marked by long open states (downward deflections). B. The frequency of long openings in the presence of aldosterone can be decreased by pre-treating cells with inhibitors of Ki-Ras. C. This maneuver also leads to the shortening of openings.
Molecular biology

Idealized schematic showing one way that we test the molecular basis for modulation of gene expression by aldosterone. Two reporter constructs are challenged with aldosterone. Only the one containing the aldosterone-responsive DNA segment respond to hormonal treatment with increased expression of luciferase.

 

 

Lab Members

 

Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow
 Nina Boiko, Ph.D.
Research Scientist

Publications

Toney GM, Vallon V, Stockand JDIntrinsic control of sodium excretion in the distal nephron by inhibitory purinergic regulation of the epithelial Na(+) channel. Curr Opin Nephrol Hypertens. 2012 Jan;21(1):52-60. Review. PMID: 22143248 [PubMed – indexed for MEDLINE

Mironova E, Peti-Peterdi J, Bugaj V, Stockand JDDiminished paracrine regulation of the epithelial Na+ channel by purinergic signaling in mice lacking connexin 30. J Biol Chem. 2011 Jan 14;286(2):1054-60. Epub 2010 Nov 12. PMID: 21075848 [PubMed – indexed for MEDLINE]

 

Kucher V, Boiko N, Pochynyuk O, Stockand JDVoltage-dependent gating underlies loss of ENaC function in Pseudohypoaldosteronism type 1. Biophys J. 2011 Apr 20;100(8):1930-9. PMID: 21504729 [PubMed – indexed for MEDLINE]

 

Bugaj V, Mironova E, Kohan DE, Stockand JDCollecting duct-specific endothelin B receptor knockout increases ENaC activity. Am J Physiol Cell Physiol. 2012 Jan;302(1):C188-94. Epub 2011 Sep 14. PMID: 21918182 [PubMed – indexed for MEDLINE]

Pochynyuk O, Rieg T, Bugaj V, Schroth J, Fridman A, Boss GR, Insel PA, Stockand JD, Vallon V.Dietary Na+ inhibits the open probability of the epithelial sodium channel in the kidney by enhancing apical P2Y2-receptor tone. FASEB J. 2010 Jan 22.

Bugaj V, Pochynyuk O, Stockand JDActivation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption. Am J Physiol Renal Physiol. 2009 Nov;297(5):F1411-8.

Dorofeeva NA, Karpushev AV, Nikolaev MV, Bolshakov KV, Stockand JD, Staruschenko A. Muscarinic M1 modulation of acid-sensing ion channels.Neuroreport. 2009 Sep 1.

Pochynyuk O, Bugaj V, Vandewalle A, Stockand JDPurinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells. Am J Physiol Renal Physiol. 2008 Jan;294(1):F38-46

Stockand JD, Staruschenko A, Pochynyuk O, Booth RE, Silverthorn DU. Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure. IUBMB Life. 2008 Sep;60(9):620-8

Bugaj V, Pochynyuk O, Mironova E, Vandewalle A, Medina JL, Stockand JDRegulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct. Am J Physiol Renal Physiol. 2008 Oct;295(4):F1063-70.

Pochynyuk O, Bugaj V, Stockand JDPhysiologic regulation of the epithelial sodium channel by phosphatidylinositidesCurr Opin Nephrol Hypertens. 2008 Sep;17(5):533-40.

Pochynyuk O, Bugaj V, Rieg T, Insel PA, Mironova E, Vallon V, Stockand JDParacrine regulation of the epithelial Na+ channel in the mammalian collecting duct by purinergic P2Y2 receptor tone.
J Biol Chem. 2008 Dec 26;283(52):36599-607.

Staruschenko A, Pochynyuk O, Vandewalle A, Bugaj V, Stockand JDAcute Regulation of the Epithelial Na+ Channel by Phosphatidylinositide 3-OH Kinase Signaling in Native Collecting Duct Principal Cells. J Am Soc Nephrol. 2007 Jun;18(6):1652-61.

Staruschenko A, Dorofeeva NA, Bolshakov KV, Stockand JDSubunit-dependent cadmium and nickel inhibition of acid-sensing ion channels. Dev Neurobiol. 2007 Jan;67(1):97-107.

Pochynyuk O, Tong Q, Medina J, Vandewalle A, Staruschenko A, Bugaj V, Stockand JDMolecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel. J Gen Physiol. 2007 Oct;130(4):399-413.

Pochynyuk O, Stockand JD, Staruschenko A. Ion channel regulation by Ras, Rho, and Rab small GTPases.Exp Biol Med (Maywood). 2007 Nov;232(10):1258-65. Review

Staruschenko A, Booth RE, Pochynyuk O, Stockand JD, Tong Q. Functional reconstitution of the human epithelial Na+ channel in a mammalian expression system. Methods Mol Biol. 2006;337:3-13.

Staruschenko A, Dorofeeva NA, Bolshakov KV, Stockand JDSubunit-dependent cadmium and nickel inhibition of acid-sensing ion channels. J Neurobiol. 2006 Dec 1;67(1):97-107.

Pochynyuk O, Tong Q, Staruschenko A, Stockand JDBinding and direct activation of the epithelial Na+ channel 

Pochynyuk O, Tong Q, Staruschenko A, Ma HP, Stockand JD.  Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides.  Am J Physiol Renal Physiol. 2006 May;290(5):F949-57.

Pochynyuk O, Medina J, Gamper N, Genth H, Stockand JD, Staruschenko A. Rapid translocation and insertion of the epithelial Na+ channel in response to RhoA signaling.  J Biol Chem. 2006 Sep 8;281(36):26520-7.

Tong Q, and Stockand JDReceptor tyrosine kinases mediate epithelial Na(+) channel inhibition by epidermal growth factor.  Am J Physiol Renal Physiol. 2005 Jan;288(1):F150-61.

Stockand JDPreserving salt: in vivo studies with Sgk1-deficient mice define a modern role for this ancient protein. Am J Physiol Regul Integr Comp Physiol. 2005 Jan;288(1):R1-3.

Gamper N, Stockand JD, and Shapiro MS. The use of Chinese hamster ovary (CHO) cells in the study of ion channels. J Pharmacol Toxicol Methods. 2005 May-Jun;51(3):177-85.

 

Staruschenko A, Pochynyuk OM, Tong Q, and Stockand JDRas couples phosphoinositide 3-OH kinase to the epithelial Na+ channel. Biochim Biophys Acta. 2005 May 20;1669(2):108-15.

Staruschenko A, Adams E, Booth RE, and Stockand JDEpithelial Na+ channel subunit stoichiometry.Biophys J. 2005 Jun;88(6):3966-75.

Tong Q, Menon AG, and Stockand JDFunctional polymorphisms in the {alpha}-subunit of the human epithelial Na+ channel increase activity. Am J Physiol Renal Physiol. 2005 Oct 25.

Pochynyuk O, Staruschenko A, Tong Q, Medina J, and Stockand JDIdentification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channelBiol Chem. 2005 Nov 11;280(45):37565-71.

Staruschenko A, Pochynyuk O, and Stockand JD.Regulation of epithelial Na+ channel activity by conserved serine/threonine switches within sorting signals. J Biol Chem. 2005 Nov 25;280(47):39161-7.

Bhalla V, Daidie D, Li H, Pao AC, LaGrange LP, Wang J, Vandewalle A, Stockand JD, Staub O,and Pearce D. Serum- and glucocorticoid-regulated kinase 1 regulates ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 by inducing interaction with 14-3-3. Mol Endocrinol. 2005 Dec;19(12):3073-84

Staruschenko A, Nichols A, Medina JL, Camacho P, Zheleznova NN, and Stockand JDRho small GTPases activate the epithelial Na(+) channel. J Biol Chem. 2004 Nov 26;279(48):49989-94.