A Region Directly Following the Second Transmembrane Domain in γENaC Is Required for Normal Channel Gating

Booth, Rachell E.; Tong, Qiusheng; Medina, Jorge; Snyder, Peter M.; Patel, P. Y.; Stockand, James D.

doi:10.1074/jbc.m305400200

Cited by 22 publications

(47 citation statements)

References 54 publications

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“…This complex is reduced by dithiothreitol and lithium dodecyl sulfate to monomers, which have molecular masses near 90 kDa. This mass for monomers is similar to what we have reported previously for ENaC subunits expressed in CHO cells (16).…”

Section: Resultssupporting

confidence: 76%

“…We believe this not to be the case, because we see no consistent channel clustering when assaying ENaC reconstituted in CHO cells at the single channel level (16,17). In addition, this notion is not well supported in the literature (8,29,30), though, Jovov and colleagues (31) have suggested that ENaC expressed in planar lipid bilayers and Xenopus laevis oocytes may cluster in an actin-dependent manner to form a channel with distinct biophysical properties.…”

Section: Discussionmentioning

confidence: 64%

“…Initially, channel subunit cDNAs were amplified by standard PCR from the respective pCMV-Myc-ENaC plasmids described previously by our laboratory (16). For ␣-, ␤-, and ␥ENaC, the upstream and downstream primers were 5Ј-CGAACTCGAGTTATGCTGGACCACAC-CAGAGC and 5Ј-GCTAGAATTCTCAGAGTGCCATGGCCGGAGC; 5Ј-CGAACTCGAGTTATGCCAGTGAAGAAGTACC and 5Ј-GCTAGAATT-CCTAGATGGCCTCCACCTCACTG; and 5Ј-CGAACTCGAGTTATGG-CGCCTGGAGAGAAG and 5Ј-GCTAGAATTCTTAGAACTCATTGGTC-AACTG, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation experiments followed published protocols (16,18,20). In brief, cells were co-transfected with Myc-and HA-tagged, as well as fluorophoretagged ENaC subunits in such a manner that similar subunits contained either a Myc or HA tag and the other two subunits contained an eYFP tag (i.e.…”

Section: Methodsmentioning

confidence: 99%

“…Protein was separated by PAGE in NuPAGE running buffer at 30 V for 24 h at 4°C and then subsequently electroblotted to nitrocellulose. Blots were probed with anti-Myc antibody as described previously (16,17).…”

Section: Methodsmentioning

confidence: 99%

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Fluorescence Resonance Energy Transfer Analysis of Subunit Stoichiometry of the Epithelial Na+ Channel

Staruschenko

Medina

Patel

et al. 2004

Journal of Biological Chemistry

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Activity of the epithelial Na ؉ channel (ENaC) is ratelimiting for Na ؉ (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, ␣, ␤, and ␥, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2␣:1␤:1␥ stoichiometry and a higher ordered channel with a 3␣:3␤:3␥ stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of ϳ90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3␣:3␤:3␥ stoichiometry.Ion channels are integral membrane proteins that form selective pores in the plasma membrane through which ions move down their electrochemical gradients. The epithelial Na ϩ channel (ENaC) 1 is a member of the ENaC/Deg gene superfamily (1-4). This family contains several ion channels (e.g. ASIC, BNaC, DEG, DRASIC, ENaC, and FaNaCH) of similar structure that serve diverse physiological functions from modulating mechanosensory transduction and neuronal signaling to control of electrolyte movement across epithelial barriers.Functional ENaC, similar to most other ion channels, is a heteromeric protein complex containing several distinct channel subunits. Four ENaC subunits have been identified, ␣, ␤, ␥, and ␦ (1, 5, 6). The first three subunits are widely expressed and together form the channel resident to the luminal plasma membrane of epithelial cells capable of electrogenic Na ϩ (re-)absorption. Consistent with this idea are findings showing that co-expression of only two of these three subunits produces little to no current in heterologous expression systems (1,5,7,8). The ␦ENaC subunit localizes to the brain and reproductive tissues where it is believed to substitute for ␣ENaC in the functional channel (6). Importantly, the stoichiometric relationship between ENaC subunits is uncertain.Each ENaC subunit has a cytosolic NH 2 and COOH terminus with two transmembrane domains separated by a single large extracellular loop with all three ENaC subunits, ␣, ␤, and ␥, contributing the functional pore (1, 5, 9, 10). Several laboratories have tested ENaC subunit stoichiometry with a biophysical approach ultimately using MacKinnon's formulation to est...

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Section: Resultssupporting

confidence: 76%

Section: Discussionmentioning

confidence: 64%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Fluorescence Resonance Energy Transfer Analysis of Subunit Stoichiometry of the Epithelial Na+ Channel

Staruschenko

Medina

Patel

et al. 2004

Journal of Biological Chemistry

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Physiology of Endothelin and the Kidney

Kohan

Inscho

Wesson

et al. 2011

Comprehensive Physiology

104

136

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Since its discovery in 1988 as an endothelial cell-derived peptide that exerts the most potent vasoconstriction of any known endogenous compound, endothelin (ET) has emerged as an important regulator of renal physiology and pathophysiology. This review focuses on how the ET system impacts renal function in health; it is apparent that ET regulates multiple aspects of kidney function. These include modulation of glomerular filtration rate and renal blood flow, control of renin release, and regulation of transport of sodium, water, protons and bicarbonate. These effects are exerted through ET interactions with almost every cell type in the kidney, including mesangial cells, podocytes, endothelium, vascular smooth muscle, every section of the nephron, and renal nerves. In addition, while not the subject of the current review, ET can also indirectly affect renal function through modulation of extrarenal systems, including the vasculature, nervous system, adrenal gland, circulating hormones and the heart. As will become apparent, these pleiotropic effects of ET are of fundamental physiologic importance in the control of renal function in health. In addition, to help put these effects into perspective, we will also discuss, albeit to a relatively limited extent, how alterations in the ET system can contribute to hypertension and kidney disease.

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