Rescue of dysfunctional deltaF508-CFTR chloride channel activity by IBMX.
Nucleotide-dependent gating of DeltaF508-CFTR was evaluated in membrane patches excised from HEK 293 and mouse L-cells and compared to observations on wt-CFTR channels recorded in the same expression systems. DeltaF508-CFTR exhibited PKA activated, ATP-dependent channel gating. When compared to wt-CFTR, the Km for ATP was increased by ninefold (260 micron vs. 28 micron) and maximal open probability (Po) was reduced by 49% (0.21 +/- 0.06 vs. 0.41 +/- 0. 02). Additionally, in the absence of PKA, DeltaF508-CFTR inactivated over a 1 to 5 min period whereas wt-CFTR remained active. Time-dependent inactivation could be mimicked in wt-CFTR by the intermittent absence of ATP in the cytosolic solution. The effects of 3-isobutyl-1-methyl xanthine (IBMX), a compound reported to stimulate DeltaF508-CFTR, were evaluated on wt- and DeltaF508-CFTR channels. At concentrations up to 5 mm, IBMX caused a concentration dependent reduction in the observed single channel amplitude (i) of wt-CFTR (maximal observed reduction 35 +/- 3%). However, IBMX failed to significantly alter total patch current because of a concomitant 30% increase in Po. The effects of IBMX on DeltaF508-CFTR were similar to effects on wt-CFTR in that i was reduced and Po was increased by similar magnitudes. Additionally, DeltaF508-CFTR channel inactivation was dramatically slowed by IBMX. These results suggest that IBMX interacts with the ATP-bound open state of CFTR to introduce a short-lived nonconducting state which prolongs burst duration and reduces apparent single channel amplitude. A secondary effect observed in DeltaF508-CFTR, which may result from this interaction, is a prolongation of the activated state. In light of previously proposed linear kinetic models of CFTR gating, these results suggest that IBMX traps CFTR in an ATP-bound state which may preclude inactivation of DeltaF508-CFTR.
Schultz
B D
BD
Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA.
Frizzell
R A
RA
Bridges
R J
RJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D004553:Electric Conductivity; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation
C505032:CFTR protein, human; D018118:Chloride Channels; D009711:Nucleotides; D010726:Phosphodiesterase Inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D015056:1-Methyl-3-isobutylxanthine