Preventing Thermal Inactivation of ΔF508-CFTR Channel Activity by NBD1 Suppressor Mutations and by Constitutively Active Intracellular Loop Mutations

ΔF508 is a temperature sensitive CFTR mutant that causes cystic fibrosis by disrupting CFTR processing, trafficking and channel gating. To address the question of thermal stability of ΔF508-CFTR channel gating, we examined WT- and ΔF508-CFTR channel activity in excised patches at varying temperature (23 to 36°C) with or without exposure to CFTR correctors and potentiators. In addition, constructs that combined the ΔF508 mutation with intracellular loop mutations that promote ATP-independent channel gating and with NBD1 suppressor mutations that enhance ΔF508-CFTR biosynthetic maturation were examined under the same conditions. ΔF508-CFTR showed a transient increase in channel activity upon increasing the temperature to 36°C followed by an irreversible current decrease with a time constant of 212 ±15 sec (n=10). Temperature induced-current decrease was more than 90% of control current at 23°C. Several CFTR correctors and potentiators promoted ΔF508-CFTR surface expression or channel activity at 23°C, but all failed to stabilize ΔF508-CFTR channel activity at 36°C. CFTR suppressor mutations corrected biosynthetic maturation of ΔF508-CFTR at 37°C and restored channel activity at 23°C as compared with WT-CFTR. In addition, these suppresser mutations completely prevented current rundown of ΔF508-CFTR at 36°C, indicating they structurally corrected ΔF508-CFTR. Constitutive loop mutations (K978C and K190C) were unable to promote the biosynthetic maturation of ΔF508-CFTR, but greatly increased ΔF508-CFTR channel activity at 23°C in large part by increasing ATP-independent channel activity. Interestingly, the ATP-independent channel activities of the combination ΔF508/loop mutants were largely resistant to thermal inactivation, whereas the ATP-dependent activities of these constructs virtually disappeared at 36°C. Our results indicate that ΔF508-CFTR gating is thermally labile and ATP-independent channel gating of ΔF508 mutant channels is more thermally stable and may be a potential target for CF drug development.