Molecular and functional characterization of the cystic fibrosis transmembrane conductance regulator from the Australian common brushtail possum, Trichosurus vulpecula.
Unlike eutherian mammals, the colon of the Australian common brushtail possum, Trichosurus vulpecula, a metatherian mammal, is incapable of electrogenic Cl(-) secretion and has elevated levels of electrogenic Na(+) absorption, while the ileum secretes HCO (3) (-) rather than Cl(-). In eutherian mammals, the cystic fibrosis transmembrane conductance regulator (CFTR) is essential for both Cl(-) and HCO (3) (-) secretion and the regulation of Na(+) absorption. Therefore, we have sequenced possum (p)CFTR, described its distribution and characterized the properties of cloned pCFTR expressed in Fischer rat thyroid (FRT) cells. pCFTR (GenBank accession No. AY916796) has a 1,478 amino acid open reading frame, which has >90% identity with CFTR from other marsupials and >80% identity with non-rodent eutherian mammals. In pCFTR, there is a high level of conservation of the transmembrane and nucleotide binding domains although, with the exception of other marsupials, there is considerable divergence from other species in the R domain. FRT cells transfected with pCFTR express mature CFTR protein which functions as a small Cl(-) channel activated by cAMP-dependent phosphorylation. In whole-cell recordings it has a linear, time and voltage-independent conductance, with a selectivity sequence P(Br) > P(Cl) > P(I) > P(HCO)(3) >> P(Gluconate). pCFTR transcript is present in a range of epithelia, including the ileum and the colon. The presence of pCFTR in the ileum and its measured HCO (3) (-) permeability suggest that it may be involved in ileal HCO (3) (-) secretion. Why the possum colon does not secrete Cl(-) and has elevated electrogenic Na(+) absorption, despite the apparent expression of CFTR, remains to be determined.
Demmers
K J
KJ
AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel 9024, New Zealand.
Carter
D
D
Fan
S
S
Mao
P
P
Maqbool
N J
NJ
McLeod
B J
BJ
Bartolo
R
R
Butt
A G
AG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001639:Bicarbonates / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D007413:Intestinal Mucosa / Q000378:metabolism*; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010802:Phylogeny; D051381:Rats; D011916:Rats, Inbred F344; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013045:Species Specificity; D013961:Thyroid Gland / Q000378:metabolism; D048330:Trichosurus
D001639:Bicarbonates; D002712:Chlorides; D017931:DNA Primers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator