External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.
Cantiello
H F
HF
Renal Unit, Massachusetts General Hospital, Boston 02114.
Prat
A G
AG
Reisin
I L
IL
Ercole
L B
LB
Abraham
E H
EH
Amara
J F
JF
Gregory
R J
RJ
Ausiello
D A
DA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental; D008565:Membrane Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D013237:Stereoisomerism; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology
C505032:CFTR protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015124:8-Bromo Cyclic Adenosine Monophosphate; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; C022570:adenosine 5'-O-(2-thiotriphosphate); D000968:Antimycin A; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D014544:Uridine Triphosphate