Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel.
A key protein component of the amiloride-sensitive sodium channel has been cloned from rat colon and human lung. It may represent the first member of a new family of ionic channels expressed from nematode to human. The biochemical properties of the rat protein, a 699 amino acids long polypeptide, have been analyzed. Four polyclonal antibodies raised against distinct parts of the channel immunoprecipitated a glycosylated protein of 96 kDa after cRNA expression in oocytes as well as after in vitro translation. When expressed alone into oocytes, the protein was not stable; most of it remains stacked into the endoplasmic reticulum. This results in a very low yield of complete maturation of the protein at the cell surface after expression from the pure cRNA. To determine the membrane topology of the protein, in vitro translation by a rabbit reticulocyte lysate was performed followed by insertion into canine pancreatic microsomes and protease digestion. Analysis revealed a model with only two transmembrane alpha helices and a large extracellular domain of about 500 amino acids. The NH2 and COOH termini are cytoplasmic. Protease digestion results suggest the possible presence of a structural element that could have a function similar to that of the H5 segment in K+ channels. The model indicates that there is no cytoplasmic site for protein kinase A phosphorylation. The well known regulation of the channel activity by hormones that activate this kinase such as vasopressin might thus be situated on another channel component.
Renard
S
S
Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, Valbonne, France.
Lingueglia
E
E
Voilley
N
N
Lazdunski
M
M
Barbry
P
P
D000584:Amiloride / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry* / Q000187:drug effects; D014981:Xenopus
D015222:Sodium Channels; D000584:Amiloride