Rescue of dysfunctional ΔF508-CFTR chloride channel activity by IBMX

Nucleotide-dependent gating of Delta wF508-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. Delta F508-CFTR exhibited PKA activated, ATP-dependent channel gating. When compared to wt-CFTR, the K-m for ATP was increased by ninefold (260 mu M vs. 28 mu M) and maximal open probability (P-o) was reduced by 49% (0.21 +/- 0.06 vs. 0.41 +/- 0.02). Additionally, in the absence of PKA, Delta F508-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 Delta F508-CFTR, were evaluated on wt- and Delta F508-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 P-o. The effects of IBMX on Delta F508-CFTR were similar to effects on wt-CFTR in that i was reduced and P-o was increased by similar magnitudes. Additionally, Delta F508-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 Delta F508-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 Delta F508-CFTR.