Cloning and functional studies of splice variants  of the α-subunit of the amiloride-sensitive Na+ channel

The α-subunit of the amiloride-sensitive epithelial Na+ channel (αENaC) is critical in forming an ion conductive pore in the membrane. We have identified the wild-type and three splice variants of the human αENaC (hαENaC) from the human lung cell line H441, using RT-PCR. These splice variants contain various structures in the extracellular domain, resulting in premature truncation (hαENaCx), 19-amino acid deletion (hαENaC−19), and 22-amino acid insertion (hαENaC+22). Wild-type hαENaC and splice variants were functionally characterized in Xenopus oocytes by coexpression with hENaC β- and γ-subunits. Unlike wild-type hαENaC, undetectable or substantially reduced amiloride-sensitive currents were observed in oocytes expressing these splice variants. Wild-type hαENaC was the most abundantly expressed hαENaC mRNA species in all tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and functional diversity of hαENaC and that alternative splicing may play a role in regulating hENaC activity.

Download Full-text

Functional polymorphisms in the α-subunit of the human epithelial Na+ channel increase activity

AJP Renal Physiology ◽

10.1152/ajprenal.00312.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. F821-F827 ◽

Cited By ~ 25

Author(s):

Qiusheng Tong ◽

Anil G. Menon ◽

James D. Stockand

Keyword(s):

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Severe Form ◽

Na Channel ◽

Channel Activity ◽

Wild Type ◽

Α Subunit ◽

Ovary Cells ◽

Functional Polymorphisms ◽

Black Populations

Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Gain-of-function mutations in ENaC cause Liddle's syndrome: a severe form of inheritable hypertension. Several polymorphisms in α-hENaC possibly associated with abnormal Na+ handling by the kidney and the salt-sensitive hypertension prevalent in black populations have been reported. The functional effects of α-hENaC polymorphisms on channel activity, however, remain controversial and have not been directly tested in a mammalian background. We ask here whether polymorphisms at positions 334, 618, and 663 in α-hENaC influence channel activity. Activity of wild-type (A334, C618, A663) and polymorphic ENaC expressed in Chinese hamster ovary cells was assessed with patch-clamp electrophysiology. While the A334T polymorphism had little effect on macroscopic ENaC currents, the C618F and A663T polymorphisms significantly increased ENaC activity >3.3- and 1.6-fold, respectively. Similarly, polymorphic ENaC had greater activity compared with wild-type channels in excised patches with activity of C618F and A663T channels increased 3.8- and 2.6-fold, respectively. Unitary channel conductances and reversal potentials were not different for polymorphic and wild-type ENaC. Increases in activity resulted primarily from increases in the apparent number of active (polymorphic) channels in the plasma membrane. Moreover, addition of a reducing agent to the cytosol significantly increased activity of wild-type ENaC equal to that of C618F polymorphic channels but had no effect on these latter channels. These results are consistent with the C618F and A663T polymorphisms leading to elevated ENaC activity with the possibility that they facilitate altered Na+ handling by the kidney.

Download Full-text

The Human Heart and Rat Brain IIA Na+ Channels Interact with Different Molecular Regions of the β1 Subunit

The Journal of General Physiology ◽

10.1085/jgp.20028703 ◽

2002 ◽

Vol 120 (6) ◽

pp. 887-895 ◽

Cited By ~ 31

Author(s):

Thomas Zimmer ◽

Klaus Benndorf

Keyword(s):

Rat Brain ◽

Human Heart ◽

Xenopus Oocytes ◽

Extracellular Domain ◽

Na Channel ◽

Na Channels ◽

Membrane Anchor ◽

Α Subunit ◽

Extracellular Region ◽

Recovery From Inactivation

The α subunit of voltage-gated Na+ channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory β1, but not the β2 subunit. In the present study, we used β1/β2 chimeras to identify molecular regions within the β1 subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na+ channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the β1/β2 chimeras with rat brain IIA channels. In agreement with previous studies, the β1 extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the β1 subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the β1 subunit was required. An exchange of the β1 membrane anchor by the corresponding β2 subunit region almost completely abolished the effects of the β1 subunit on hH1, suggesting that the β1 membrane anchor plays a crucial role for the modulation of the cardiac Na+ channel isoform. It is concluded that the β1 subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.

Download Full-text

Expression of the B splice variant of NBCe1 (SLC4A4) in the mouse kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00515.2017 ◽

2018 ◽

Vol 315 (3) ◽

pp. F417-F428 ◽

Cited By ~ 5

Author(s):

Lijuan Fang ◽

Hyun-Wook Lee ◽

Chao Chen ◽

Autumn N. Harris ◽

Michael F. Romero ◽

...

Keyword(s):

Splice Variant ◽

Splice Variants ◽

Immunoblot Analysis ◽

Mouse Kidney ◽

Wild Type ◽

Rt Pcr ◽

Outer Medulla ◽

Bicarbonate Transporter ◽

Outer Stripe ◽

Acid Loading

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study’s purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

Download Full-text

On the Molecular Basis of Ion Permeation in the Epithelial Na+ Channel

The Journal of General Physiology ◽

10.1085/jgp.114.1.13 ◽

1999 ◽

Vol 114 (1) ◽

pp. 13-30 ◽

Cited By ~ 94

Author(s):

Stephan Kellenberger ◽

Nicole Hoffmann-Pochon ◽

Ivan Gautschi ◽

Estelle Schneeberger ◽

Laurent Schild

Keyword(s):

Amino Acid ◽

Single Channel ◽

Kinetic Property ◽

Selectivity Filter ◽

Na Channel ◽

Amino Acid Residues ◽

Discrete State ◽

Α Subunit ◽

Α Helix ◽

Epithelial Na Channel

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two α, one β, and one γ homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of β and γ subunits, which precedes the second putative transmembrane α helix (M2). A residue in the α subunit (αS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of α, β, and γ subunits (αG587, βG529, γS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of βG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported αS589 mutations. We conclude that the residues αG587 to αS589 and homologous residues in the β and γ subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.

Download Full-text

The Atg16l1 gene: characterization of wild type, knock-in, and knock-out phenotypes in rats

Physiological Genomics ◽

10.1152/physiolgenomics.00114.2020 ◽

2021 ◽

Author(s):

Kari Chesney ◽

Hongsheng Men ◽

Miriam A Hankins ◽

Elizabeth C Bryda

Keyword(s):

Amino Acid ◽

Rat Model ◽

Splice Variants ◽

Genetic Alterations ◽

Wild Type ◽

Rat Strains ◽

Specific Expression ◽

Knock Out ◽

Caspase Cleavage ◽

Increased Susceptibility

ATG16L1 is a ubiquitous autophagy gene responsible, in part, for formation of the double-membrane bound autophagosome that delivers unwanted cellular debris and intracellular pathogens to the lysosome for degradation. A single, nonsynonymous adenine to guanine polymorphism resulting in a threonine to alanine amino acid substitution (T300A) directly preceded by a caspase cleavage site (DxxD) causes an increased susceptibility to Crohn's disease (CD) in humans. The mechanism behind this increased susceptibility is still being elucidated, however, the amino acid change caused by this point mutation results in increased ATG16L1 protein sensitivity to caspase 3-mediated cleavage. In order to generate novel rat strains carrying genetic alterations in the rat Atg16l1 gene, we first characterized the wild type rat gene. We identified four alternative splice variants with tissue-specific expression. Using CRISPR-Cas9 genome editing technology, we developed a knock-in rat model for the human ATG16L1 T300A CD risk polymorphism as well as a knock-out rat model to evaluate the role of Atg16l1 in autophagy as well as its potential effect on CD susceptibility. These are the first reported rat strains with alterations of the Atg16l1 gene. Consistent with studies of the effects of human ATG16L1 polymorphisms, models exhibit morphological abnormalities in both Paneth and goblet cells, but do not develop spontaneous intestinal permeability or inflammatory bowel disease. Analysis of the gut microbiota does not show inherent differences in bacterial composition between wild type and genetically modified animals. These Atg16l1 strains are valuable new animal models for the study of both autophagy and CD susceptibility.

Download Full-text

Role of Amino Acid Residues in Transmembrane Segments IS6 and IIS6 of the Na+Channel α Subunit in Voltage-dependent Gating and Drug Block

Journal of Biological Chemistry ◽

10.1074/jbc.m206126200 ◽

2002 ◽

Vol 277 (38) ◽

pp. 35393-35401 ◽

Cited By ~ 163

Author(s):

Vladimir Yarov-Yarovoy ◽

Jancy C. McPhee ◽

Diane Idsvoog ◽

Caroline Pate ◽

Todd Scheuer ◽

...

Keyword(s):

Amino Acid ◽

Na Channel ◽

Amino Acid Residues ◽

Α Subunit ◽

Transmembrane Segments ◽

Voltage Dependent

Download Full-text

Na transport proteins are expressed by rat alveolar epithelial type I cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00130.2000 ◽

2002 ◽

Vol 282 (4) ◽

pp. L599-L608 ◽

Cited By ~ 98

Author(s):

Zea Borok ◽

Janice M. Liebler ◽

Richard L. Lubman ◽

Martha J. Foster ◽

Beiyun Zhou ◽

...

Keyword(s):

Alveolar Epithelial Cells ◽

Na Channel ◽

Type I ◽

Rt Pcr ◽

Na Transport ◽

Double Labeling ◽

Α Subunit ◽

Alveolar Epithelial ◽

Na Pump

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the α-subunit of the rat (r) epithelial Na channel (α-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump α1-subunit in AEC was assessed in situ. Na pump subunit isoform and α-rENaC expression was also evaluated by RT-PCR in highly purified (∼95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-α1 and anti-β1 Na pump subunit and anti-α-rENaC antibodies was detected, while reactivity with anti-α2 Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-α1 Na pump subunit antibody. Na pump α1- and β1- (but not α2-) subunits and α-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.

Download Full-text

Cloning and Characterization of the Zebrafish mll Ortholog.

Blood ◽

10.1182/blood.v110.11.1249.1249 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1249-1249

Author(s):

Blaine W. Robinson ◽

Giuseppe Germano ◽

Yuanquan Song ◽

Rita J. Balice-Gordon ◽

Carolyn A. Felix

Keyword(s):

Amino Acid ◽

Blot Analysis ◽

Temporal Pattern ◽

Southern Blot Analysis ◽

Early Embryogenesis ◽

Amino Acid Sequences ◽

Pcr Analysis ◽

Adult Zebrafish ◽

Wild Type ◽

Rt Pcr

Abstract Introduction: Zebrafish enable studies of early embryogenesis and hematopoiesis like no other animal models. Many zebrafish orthologs of hematopoietic genes have been identified, and zebrafish models of leukemia are emerging but the zebrafish ortholog of MLL, a critical oncogene disrupted by leukemogenic translocations, has not yet been studied. We cloned the complete zmll cDNA and characterized its temporal expression as a framework for further studies of MLL where current knowledge is still incomplete. Methods: Bioinformatic tools were employed to interrogate the existence and relationship of a zmll ortholog and synteny with human MLL. Degenerate RT-PCR was used to determine whether MLL amino acid sequences in domains highly conserved across species could identify the orthologous zebrafish transcript. Cross-species Southern blot analysis was performed to determine if a predicted zmll gene from restriction map simulations of a projected genomic sequence could be detected with a human cDNA probe for the MLL breakpoint cluster region (bcr). The full-length zmll cDNA was obtained using a combination of 5′ RACE PCR, long-range and conventional PCR. The corresponding protein was analyzed in a phylogram tree. The temporal pattern of zmll RNA expression was examined using quantitative (Q) RT-PCR. Results: Bioinformatic analysis using the human MLL protein as the reference sequence identified two putative “similar to MLL proteins” and predicted two proximal transcript sequences on zebrafish chromosome 15. Gene prediction tools suggested a single genomic structure matching both protein sequences. A conserved block of synteny containing several linked genes surrounded the predicted zmll. Furthermore, zmll and human MLL were in the same map order in an uninterrupted segment with the gene for ubiquitination factor E4A. Degenerate RT-PCR analysis of wild-type adult zebrafish RNA based on cross-species amino acid sequences from highly conserved PHD and SET domains amplified the predicted transcript. Cross-species Southern blot analysis with the human probe detected the projected zmll genomic fragments corresponding to the MLL bcr in adult zebrafish genomic DNA. RT-PCR analysis of wild-type adult zebrafish RNA determined that the two predicted “similar to MLL proteins” were from a single gene. The full length 12657 bp, 35-exon zmll cDNA cloned from wild-type 24 hpf zebrafish embryo RNA predicted a 4218 amino acid protein with CXXC, PHD, Bromodomain, FYRN, FYRC, and SET domains and taspase cleavage sites with 45.8% sequence identity and 57.3% similarity to human MLL. Phylogram tree analysis suggested evolutionary divergence of mammals from teleosts but nonetheless conservation of critical functional domains. QRT-PCR demonstrated maternally supplied zmll mRNA during the earliest embryonic developmental timepoints, and expression of zygotic zmll mRNA during embryogenesis and in the zebrafish adult. Conclusions: These results indicate that there is a single zmll gene with highly conserved functional similarity to human MLL. The temporal pattern of expression, including maternal supply of transcript to the embryo, indicates that zmll is important from early embryogenesis through the entire lifespan of the fish. The high evolutionary conservation of critical domains creates the framework to use zebrafish for studying MLL in hematopoiesis and leukemia.

Download Full-text

Lysine residues 2 and 104 of the human chorionic gonadotrophin β subunit influence receptor binding

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0100337 ◽

1993 ◽

Vol 10 (3) ◽

pp. 337-343 ◽

Cited By ~ 14

Author(s):

H Xia ◽

J Huang ◽

T-M Chen ◽

D Puett

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Basic Amino Acid ◽

Chorionic Gonadotrophin ◽

Human Chorionic Gonadotrophin ◽

Amino Acid Residues ◽

Β Subunit ◽

Wild Type ◽

Α Subunit ◽

Lysine Residues

ABSTRACT Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu2) and to hCG-β (Glu104), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.

Download Full-text