Thermally Unstable Gating of the Most Common Cystic Fibrosis Mutant Channel (ΔF508)

摘要

Most cystic fibrosis (CF) cases are caused by the ΔF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of ΔF508-CFTR can be “rescued” by culturing cells at 26–28 °C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a ΔF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ΔF508-CFTR function. Low temperature-rescued ΔF508-CFTR channels irreversibly inactivated with a time constant of 5–6 min when excised patches were warmed from 22 °C to 36.5 °C. A panel of CFTR correctors and potentiators that increased ΔF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued ΔF508-CFTR maturation and stabilized channel activity at 36.5 °C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued ΔF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these ΔF508-CFTR constructs were stable at 36.5 °C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) ΔF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from ΔF508-induced thermal instability, a finding that may inform alternative treatment strategies.