The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients

Abstract

Background: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown.

Methods: We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion.

Results: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca²⁺-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl⁻ conductance.

Conclusions: We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

Figure 1. 1-EBIO activates CFTR-mediated basal and cholinergic Cl⁻ secretion in human rectal biopsies.

(A,B) Original recordings of effects of 1-EBIO (500 µM, basolateral) on basal and carbachol-induced (CCH) transepithelial voltage (V_te) and transepithelial resistance (R_te) across rectal biopsies from a control subject (A) and a CF patient carrying two severe CFTR mutations (R1158X/2183AA>G). (B) Experiments were performed in the presence of amiloride and indomethacin. Lumen-positive V_te responses reflect K⁺ secretion and lumen-negative responses reflect Cl⁻ secretion. R_te was determined from V_te downward deflections obtained by pulsed current injection. (C) Summary of effects of 1-EBIO on basal equivalent short-circuit current (I_sc') in rectal biopsies from control subjects and CF patients with no detectable Cl⁻ secretion (CF_absent). (D,E) Effects of 1-EBIO on CCH-induced peak (open bars) and plateau (closed bars) I_sc' responses in control (D) and CF_absent rectal tissues (E). (F,G) Effect of CFTR_inh-172 on 1-EBIO-induced Cl⁻ secretion (lumen-negative I_sc') under basal conditions (F) and on carbachol-induced (CCH) Cl⁻ secretion in the presence of 1-EBIO (G) in rectal biopsies from control subjects. Data are presented as mean±SEM. n = 7–13 individuals per group. * P<0.001 and ^† P<0.01.

Figure 2. 1-EBIO potentiates cAMP-mediated and cholinergic Cl secretion in human rectal biopsies and this effect is abrogated by inhibition of Ca²⁺-dependent K⁺ channels with clotrimazole.

(A) Original recording of effects of 1-EBIO (500 µM, basolateral) on cAMP-induced Cl⁻ secretion (IBMX/forskolin) and cholinergic co-activation (CCH), and effects of clotrimazole (30 µM, basolateral) on Cl⁻ secretory responses in a rectal biopsy from a control subject. Experiments were performed in the presence of amiloride, indomethacin and IBMX/forskolin. (B) Summary of effects of 1-EBIO on cAMP-induced Cl⁻ secretion and inhibition by clotrimazole in rectal tissues from control subjects. (C) Concentration-response curve for 1-EBIO-induced Cl⁻ secretion was determined in the presence of cAMP-mediated activation (IBMX/forskolin). (D) Effects of 1-EBIO on CCH-induced Cl⁻ secretion in the presence of IBMX/forskolin and inhibition by clotrimazole in control rectal tissues. Data are presented as mean±SEM. n = 17 individuals per group. *P<0.001. (E) RT-PCR analysis revealed transcripts of the clotrimazole-sensitive Ca²⁺-activated K⁺ channel KCNN4 in rectal biopsies from control and CF subjects. The 405 bp KCNN4 fragment was only identified in the presence (+), but not in the absence of reverse transcriptase (-).

Figure 3. 1-EBIO mediated augmentation of cAMP-induced and cholinergic Cl⁻ secretion in human rectal biopsies does not depend on 293B-sensitive cAMP-dependent K⁺ channels.

(A) Original recording of effects of 1-EBIO (500 µM, basolateral) on cAMP-induced Cl⁻ secretion (IBMX/forskolin) and cholinergic co-activation (CCH), and effects of 293B (10 µM, basolateral) on Cl⁻ secretory responses in a rectal biopsy from a control subject. Experiments were performed in the presence of amiloride, indomethacin and IBMX/forskolin. (B, C) Summary of effects of 1-EBIO on cAMP-induced (B) and CCH-induced Cl⁻ secretion (C) in the absence and presence of 293B in rectal tissues from control subjects. Data are presented as mean±SEM. n = 19 individuals per group. *P<0.001. (D) RT-PCR analysis detected transcripts of the 293B-sensitive K⁺ channel KCNQ1 (728 bp fragment) in the presence (+), but not in the absence of reverse transcriptase (-), in rectal biopsies from control and CF subjects.

Figure 4. 1-EBIO potentiates residual CFTR-mediated Cl⁻ secretion in CF rectal biopsies.

(A–C) Original recordings of effects of cAMP-mediated (IBMX/forskolin) and cholinergic (CCH) activation, and effects of 1-EBIO (500 µM, basolateral) on transepithelial voltage (V_te) and resistance (R_te) in rectal tissues from a control subject (A), a CF patient with no detectable Cl⁻ secretion (CF_absent; R1158X/2183AA>G) (B), and a CF patient with residual Cl⁻ secretion (CF_residual; F508del/Y161C), as evidence by lumen-negative V_te responses (C). Experiments were performed in presence of amiloride and indomethacin. 1-EBIO potentiated cAMP-mediated and cholinergic Cl⁻ secretion in control and CF_residual rectal tissues, but did not induce Cl⁻ secretion in the CF_absent tissue.

Figure 5. 1-EBIO potentiates residual CFTR-mediated Cl⁻ secretion in CF rectal biopsies.

(A–F) Summary of effects of bumetanide (100 µM, basolateral) (A,B), CFTR_inh-172 (20 µM, basolateral) (C,D) and 1-EBIO (500 µM, basolateral) (E,F) on cAMP-mediated (IBMX/forskolin) and cholinergic (CCH) activation of equivalent short circuit current (I_sc') in rectal biopsies from control subjects, CF patients with no detectable Cl⁻ secretion (CF_absent) and CF patients with residual Cl⁻ secretion (CF_residual). All experiments were performed in the presence of amiloride and indomethacin. Only lumen-negative peak responses or plateau responses are shown for cholinergic (CCH) activation. Data are presented as mean±SEM. n = 4–26 individuals per group. *P<0.001, ^† P<0.01 and ^¶ P<0.05.