Regulation of CFTR Cl- channel gating by ADP and ATP analogues.
The cystic fibrosis gene product (CFTR) is a chloride channel which, once phosphorylated, is regulated by nucleotide phosphates (Anderson, M. P., and M. J. Welsh. 1992. Science. 257:1701-1704; Venglarik, C. J., B. D. Schultz, R. A. Frizzell, and R. J. Bridges. 1994. Journal of General Physiology. 104:123-146). Nucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFTR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 +/- 9 microM. AMP neither initiated CFTR channel gating nor inhibited ATP-dependent CFTR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP gamma S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFTR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFTR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFTR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di- or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25 degrees C, result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies.
Schultz
B D
BD
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Venglarik
C J
CJ
Bridges
R J
RJ
Frizzell
R A
RA
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006868:Hydrolysis; D015640:Ion Channel Gating; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D013696:Temperature; D013997:Time Factors
D018118:Chloride Channels; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases