A CFTR anioncsatorna extracelluláris régiójának a csatorna működésére gyakorolt hatása
Simon Márton András
Molecular Medicine
Dr. Enyedi Péter
SE Elméleti Orvostudományi Központ, Beznák Aladár terem
2023-05-24 10:00:00
Cellular and Molecular Physiology
Dr. Hunyady László
Dr. Csanády László
Dr. Varga Zoltán
Dr. HegedűsTamás
Dr. Enyedi Péter
Dr. Czirják Gábor
Dr. Mike Árpád
CFTR anion channel mutations cause the lethal and incurable disease cystic fibrosis
(CF). Gating of phosphorylated CFTR is driven by ATP binding/hydrolysis at two nucleotidebinding domains, and exhibits ‘bursting’ behavior: groups of openings (state O) separated by
short ‘flickery’ closures (state Cf) form ‘bursts’ (state B) that are flanked by long ‘interburst’
closures (state Cs). The human (hCFTR) and zebrafish (zCFTR) orthologues represent roughly
the two ends of CFTR molecular evolution and possess different gating properties and
differences in their structures. CF mutation hR117H decreases anion conductance and
accelerates pore closure but does not affect opening rate. This suggests that position 117 moves
late during the interburst-burst conformational change, and that the hR117 side chain stabilizes
the B state. In the ATP-bound phosphorylated CFTR structure the hR117 side chain forms a
strong H-bond with the hE1124 backbone carbonyl group, but that bond is absent in the ATPunbound, unphosphorylated structure.
We aimed to investigate whether the hR117–hE1124 interaction stabilizes the O state.
In non-hydrolytic backgrounds single-channel and macroscopic inside-out patch-clamp
recordings allowed quantitation of gating-associated changes in interaction energy between the
target positions through thermodynamic mutant cycles. We found that mutation hE1124Δ
accelerates closing rate and decreases intraburst open probability similarly to mutation
hR117H, but no additivity was observed in the hR117H-E1124Δ mutant. These findings reveal
that the hR117/hE1124 interaction stabilizes exclusively the O state. In the outward-facing
zCFTR structure that H-bond is not observed, and we found that zR118H mutation in zCFTR
has no functional effect. Instead, we discovered a H-bond between the zN120 and zS109 side
chains of ATP-bound phosphorylated-, but not ATP-free unphosphorylated zCFTR. Using
functional experiments, we confirmed that zS109 indeed forms a H-bond with zN120, but the
bond is formed exclusively in the Cf state. In hCFTR a bond between the analogous positions
cannot form, as an isoleucine (hI119) replaces the asparagine. Surprisingly, mutation hS108A
produces a strong hR117H-like phenotype, and the effects of these two mutations are not
additive. In conclusion, in zCFTR the zS109-zN120 interaction stabilizes the Cf state, whereas
in hCFTR the hS108-hR117-hE1124 interactions cooperate to stabilize the O state