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Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not ΔF508 CFTR from thermal instability.
Liu X
,
Dawson DC
.
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The G551D cystic fibrosis transmembrane conductance regulator (CFTR) mutation is associated with severe disease in ∼5% of cystic fibrosis patients worldwide. This amino acid substitution in NBD1 results in a CFTR chloride channel characterized by a severe gating defect that can be at least partially overcome in vitro by exposure to a CFTR potentiator. In contrast, the more common ΔF508 mutation is associated with a severe protein trafficking defect, as well as impaired channel function. Recent clinical trials demonstrated a beneficial effect of the CFTR potentiator, Ivacaftor (VX-770), on lung function of patients bearing at least one copy of G551D CFTR, but no comparable effect on ΔF508 homozygotes. This difference in efficacy was not surprising in view of the established difference in the molecular phenotypes of the two mutant channels. Recently, however, it was shown that the structural defect introduced by the deletion of F508 is associated with the thermal instability of ΔF508 CFTR channel function in vitro. This additional mutant phenotype raised the possibility that the differences in the behavior of ΔF508 and G551D CFTR, as well as the disparate efficacy of Ivacaftor, might be a reflection of the differing thermal stabilities of the two channels at 37 °C. We compared the thermal stability of G551D and ΔF508 CFTR in Xenopus oocytes in the presence and absence of CTFR potentiators. G551D CFTR exhibited a thermal instability that was comparable to that of ΔF508 CFTR. G551D CFTR, however, was protected from thermal instability by CFTR potentiators, whereas ΔF508 CFTR was not. These results suggest that the efficacy of VX-770 in patients bearing the G551D mutation is due, at least in part, to the ability of the small molecule to protect the mutant channel from thermal instability at human body temperature.
Figure 1. Thermal instability of G551D CFTR channels. (A) Following stimulation
[10 μM isoproterenol and 1 mM IBMX (hatched bar and crosshairs)],
an oocyte expressing G551D CFTR was warmed to 37 °C (gray bar
and circles) for 10 min. After recovering at 22 °C, the oocyte
was exposed to 10 μM CF172. (B) Summary of G551D CFTR conductance
before and after thermal deactivation (P < 0.05).
â represents the initial conductance following stimulation.
⡠is the minimal conductance at 37 °C. To save space,
the period prior to and during CFTR stimulation by the stimulatory
cocktail is not shown. The half-time for G551D CFTR activation averaged
37.2 ± 4 min (n = 18), and the half-time for
ÎF508 CFTR activation under comparable conditions averaged 7.0
± 2.0 min (n = 18). A prolonged half-time for
activation by cAMP is a consistent feature of G551D channels and is
compatible with the long closed times seen in single-channel recordings.24
Figure 2. Differential effects of CFTR potentiators on the thermal
instability
of G551D CFTR channels. (A) Following stimulation at 22 °C, an
oocyte expressing G551D CFTR was warmed to 37 °C (gray bar and
circles) and then exposed to 10 μM CF172 at 37 °C. Note
the break on the time axis. (BâE) Following stimulation, oocytes
expressing G551D CFTR were exposed to 10 μM VX-770, 10 μM
P2 (PG-01), 50 μM Genistein, and 10 μM P1, respectively,
warmed to 37 °C (gray bar and circles) in the presence of potentiators,
and then exposed to 10 μM CF172 at 37 °C. (F) Summary of
the ratio of conductance at the end of warming (g2) and the initial steady state conductance prior to exposure
to the potentiators (g1). Doses of the
potentiators were chosen on the basis of the maximal concentration
reported in the literature as well as our previous study.7,16,25â27 With respect
to the activated conductance prior to the application of potentiator,
the P values for stimulation by Genistein, P1, P2,
and VX770 are all <0.05.
Figure 3. VX-770 did not protect ÎF508 CFTR from thermal deactivation.
(A) Following stimulation, an oocyte expressing ÎF508 CFTR channels
was warmed to 37 °C (gray bar and circles) for 10 min. It was
then exposed to 10 μM CF172 after being cooled to 22 °C.
(B) Summary of conductance due to ÎF508 CFTR before and after
a temperature challenge (P < 0.05). (C) Following
stimulation, an oocyte expressing ÎF508 CFTR was exposed to
10 μM VX-770 and then warmed to 37 °C (gray bar and circles)
in the presence of VX-770. It was then exposed to 10 μM CF172
after being cooled to 22 °C. (D) Summary of VX-770-modified ÎF508
CFTR conductance before and after the temperature challenge (P < 0.05).
Figure 4. VX-770 stimulated G551D but not ÎF508 CFTR channels at 37
°C. (A) Following activation of conductance via an increase in
intracellular cAMP concentration (see Materials and
Methods), an oocyte expressing G551D CFTR was warmed to 37
°C (gray bar and circles). The oocyte was then exposed to 10
μM VX770 and subsequently to 10 μM CF172 at 37 °C.
(B) Following stimulation, an oocyte expressing ÎF508 CFTR was
warmed to 37 °C (gray bar and circles) and then exposed to 10
μM VX770 at 37 °C. (C) Summary of the conductance due to
G551D CFTR at 37 °C before and after stimulation by 10 μM
VX770. The CF172-insensitive conductance was subtracted from the final
analysis. (D) Summary of conductance due to ÎF508 CFTR channels
at 37 °C before and after stimulation by 10 μM VX770.
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