Biomaterial Database

Alternatively spliced forms of the alpha subunit of the epithelial sodium channel: distinct sites for amiloride binding and channel pore. 1995

X J Li, and R H Xu, and W B Guggino, and S H Snyder

Department of Neuroscience, Psychiatry, and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

The amiloride-sensitive epithelial sodium channel (ENAC) consists of at least three subunits, alpha, beta, and gamma. Sodium conductance occurs when only the alpha subunit is expressed in Xenopus oocytes, but it is greatly enhanced by coexpression of all three subunits. All three subunits have two transmembrane domains. Whether the amiloride binding site exists in the extracellular portion or a transmembrane domain has not been established. Using reverse transcription-polymerase chain reaction in rat taste tissues, we have identified two alternatively spliced transcripts of ENAC (alpha ENACa and alpha ENACb) with deletions of nucleotides that introduce a premature stop codon and may result in proteins shortened by 199 and 216 amino acids, respectively, at the carboxyl terminus. Genomic Southern blots indicate that a single gene accounts for alpha ENAC and the alternatively spliced variants. Reverse transcription-polymerase chain reaction and RNase protection assays demonstrate that alpha ENACa is expressed to a lesser extent than alpha ENAC in kidney, lung, and taste tissues. alpha ENACa differs from alpha ENAC by a deletion in the second transmembrane domain. Despite this deletion, alpha ENACa expression in transfected human embryonic kidney 293 cells or CV-1 cells augments [3H]phenamil binding. The [3H]phenamil binding of alpha ENACa resembles that of alpha ENAC, being inhibited more potently by phenamil (Kd = 65 nM) than amiloride. Unlike alpha ENAC, expression of alpha ENACa in Xenopus oocytes fails to generate amiloride-sensitive Na+ or Li+ currents. These results suggest that the amiloride binding site resides on the extracellular loop of the alpha subunit of ENAC and not the putative second transmembrane domain, which forms a channel pore. Heterogeneity in alpha ENAC isoforms may contribute to the complexity of multimeric structures and functional variation of ENAC.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009865	Oocytes	Female germ cells derived from OOGONIA and termed OOCYTES when they enter MEIOSIS. The primary oocytes begin meiosis but are arrested at the diplotene state until OVULATION at PUBERTY to give rise to haploid secondary oocytes or ova (OVUM).	Ovocytes,Oocyte,Ovocyte
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D002460	Cell Line	Established cell cultures that have the potential to propagate indefinitely.	Cell Lines,Line, Cell,Lines, Cell
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004848	Epithelium	The layers of EPITHELIAL CELLS which cover the inner and outer surfaces of the cutaneous, mucus, and serous tissues and glands of the body.	Mesothelium,Epithelial Tissue,Mesothelial Tissue,Epithelial Tissues,Mesothelial Tissues,Tissue, Epithelial,Tissue, Mesothelial,Tissues, Epithelial,Tissues, Mesothelial
D000584	Amiloride	A pyrazine compound inhibiting SODIUM reabsorption through SODIUM CHANNELS in renal EPITHELIAL CELLS. This inhibition creates a negative potential in the luminal membranes of principal cells, located in the distal convoluted tubule and collecting duct. Negative potential reduces secretion of potassium and hydrogen ions. Amiloride is used in conjunction with DIURETICS to spare POTASSIUM loss. (From Gilman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed, p705)	Amidal,Amiduret Trom,Amiloberag,Amiloride Hydrochloride,Amiloride Hydrochloride, Anhydrous,Kaluril,Midamor,Midoride,Modamide,Anhydrous Amiloride Hydrochloride,Hydrochloride, Amiloride,Hydrochloride, Anhydrous Amiloride,Trom, Amiduret
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA