Thermal Instability of ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function: Protection by Single Suppressor Mutations and Inhibiting Channel Activity

Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or Delta F508 CFTR was stable at 22 degrees C and increased at 28 degrees C, a temperature permissive for Delta F508 CFTR expression in mammalian cells. At 37 degrees C, however, CFTR-mediated conductance was further enhanced, whereas that due to Delta F508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of Delta F508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P-o) of Delta F508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P-o of Delta F508 CFTR channels at room temperature failed to protect channels from inactivation at 37 degrees C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated Delta F508CFTR channels or those inhibited by CFTRinh-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the Delta F508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.