Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein

ΔF508 CFTR surface stability is regulated by DAB2 and CHIP-mediated ubiquitination in post-endocytic compartments.

The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR

W1282X-CFTR channels exhibit exceptionally low P_os in excised HEK-293T patches under control conditions and correspondingly slow activation time courses by 300 nM VX-770.

<p>Activation conditions were identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.g001" target="_blank">Fig 1</a>. Holding potential was +60 mV throughout. Inset shows unitary currents under control activation conditions visualized at high gain. The indicated values for the control P_o, opening rate/channel and closed time constant were calculated for this control record after estimating the total number of channels in the patch from the VX-770 stimulated current. These P_o and opening rate values are maximal estimates (see Experimental Procedures). The macroscopic activation time course was fit to a single exponential with a time constant of 273s.</p

W1282X-CFTR channels in FRT patches are virtually ‘locked open’ by adding curcumin after a saturating dose of VX-770.

<p>(A) Multichannel record for inside-out patch excised from FRT cell stably expressing W1282X-CFTR. Control conditions were identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.g001" target="_blank">Fig 1</a>. 300 nM VX-770 and 30 μM curcumin were added where indicated. Holding potential = 60 mV. This patch contained 6 channels. The P_o estimated for each condition in this experiment is indicated. (B) Higher gain records for the experiment in panel A. (C) Mean P_o, single channel opening rate and mean burst duration for W1282X-CFTR channels estimated for the indicated conditions. Error bars are SEs. N’s are indicated in parentheses. *p<0.05 and **<0.01 by paired t-test as indicated.</p

W1282X-CFTR channel activation by VX-770 in HEK-293T macropatches requires PKA phosphorylation but not ATP binding.

<p>(A) Macroscopic current record showing that addition of an ATP scavenger to the bath (24U/ml hexokinase/10 mM glucose ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.ref014" target="_blank">14</a>]) did not inhibit activation of W1282X-CFTR channels by VX-770. Control conditions were identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.g001" target="_blank">Fig 1</a>. (B) Macroscopic current record showing that PKA (110 U/ml) was required for VX-770 activation of W1282X-CFTR. (C) Macroscopic current record showing detectable W1282X-CFTR current in the absence of bath PKA for a patch excised from an HEK-293T cell pre-treated with forskolin (40 μM) and IBMX (100 μM) for 5 mins before excision. 1.5 mM MgATP was present in the bath throughout this experiment. PKA (110U/ml) was added to the bath where indicated. (D) Mean basal currents measured at + 80 mV before bath PKA addition (left) and basal currents normalized to the currents measured after bath PKA addition (right) for macropatches excised from cells pre-treated (or not) with forskolin and IBMX. Errors are SEs. N’s are indicated in parentheses. ** p<0.01 compared to untreated control by unpaired t-test.</p

Curcumin and VX-770 additively stimulate W1282X-CFTR currents in excised HEK-293T macropatches.

<p>(A) Macroscopic current record showing substantial stimulation of W1282X-CFTR channels by 30 μM curcumin added to the bath prior to VX-770. This curcumin concentration was chosen because it is the maximally effective dose for stimulating Δ1198-CFTR activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.ref009" target="_blank">9</a>]. (B) Curcumin strongly stimulated W1282X-CFTR channels after they were first activated by a saturating dose of VX-770 (300 nM). (C, D) Analogous order of addition experiments for Δ1198-CFTR macropatches. (E) Mean percent stimulation of W1282X-CFTR or Δ1198-CFTR current by each compound alone normalized to the total current measured after the addition of both compounds. Errors are SEs. N’s are indicated in parentheses.</p

VX-770 robustly activates W1282X-CFTR and Δ1198-CFTR channels in excised HEK-293T macropatches.

<p>(A) Control macroscopic current record for an inside-out macropatch excised from an HEK-293T cell expressing WT-CFTR. Ramp protocol (+/- 80 mV); zero current level indicated by the dotted line. Control activation conditions were 110 U/ml PKA and 1.5 mM MgATP in the bath. VX-770 (300 nM) and a CFTR channel inhibitor, CFTR(inh)-172 (10 μM), were added to the bath where indicated. This patch contained several thousand WT-CFTR channels estimated assuming a single channel current of about 0.5 pA at + 80 mV ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.ref009" target="_blank">9</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.ref014" target="_blank">14</a>] and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152232#pone.0152232.g005" target="_blank">Fig 5</a>). VX-770 negligibly stimulated the WT-CFTR current because the channels were nearly maximally active under control condition. (B) Corresponding macroscopic record for an inside-out patch excised from an HEK-293T cell transfected with W1282X-CFTR. Conditions were identical to panel A. Note the very small control current and the marked stimulation by 300 nM VX-770. (C) Corresponding macroscopic record for patch containing Δ1198-CFTR channels. (D) Titration curve for VX-770 activation of W1282X-CFTR channels in excised macropatches. Each symbol represents the mean +/- SE averaged for 6 patches. Conditions were identical to panels A-C. Currents were normalized to the control current before drug addition. Data were fit to a one binding site model with a K_D of 10 nM. (E) Mean control currents (left) and fold stimulation by 300 nM VX-770 (right) for the indicated constructs. N’s are indicated in parentheses. Errors are SEs. The E and P versions of W1282X represent an engineered stop mutation and a patient stop mutation, respectively (see Experimental Procedures). (F) Immunoblot showing relative protein levels of the indicated constructs in transfected HEK-293T cells. Total protein loads were identical for each lane (30 μg). The upper band in each lane represents the mature, maximally glycosylated protein.</p