CFTR Cl− channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis

BACKGROUND & AIMS: Cystic fibrosis (CF) is caused by over 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and presents with a widely variable phenotype. Genotype-phenotype studies identified CFTR mutations that were associated with pancreatic sufficiency (PS). Residual Cl- channel function was shown for selected PS mutations in heterologous cells. However, the functional consequences of most CFTR mutations in native epithelia are not well established. METHODS: To elucidate the relationships between epithelial CFTR function, CFTR genotype, and patient phenotype, we measured cyclic adenosine monophosphate (cAMP)-mediated Cl- secretion in rectal biopsy specimens from 45 CF patients who had at least 1 non-DeltaF508 mutation carrying a wide spectrum of CFTR mutations. We compared CFTR genotypes and clinical manifestations of CF patients who expressed residual CFTR-mediated Cl- secretion with patients in whom Cl- secretion was absent. RESULTS: Residual anion secretion was detected in 40% of CF patients, and was associated with later disease onset (P < 0.0001), higher frequency of PS (P < 0.0001), and less severe lung disease (P < 0.05). Clinical outcomes correlated with the magnitude of residual CFTR activity, which was in the range of approximately 12%-54% of controls. CONCLUSIONS: Specific CFTR mutations confer residual CFTR function to rectal epithelia, which is related closely to a mild disease phenotype. Quantification of rectal CFTR-mediated Cl- secretion may be a sensitive test to predict the prognosis of CF disease and identify CF patients who would benefit from therapeutic strategies that would increase residual CFTR activity

The K+ Channel Opener 1-EBIO Potentiates Residual Function of Mutant CFTR in Rectal Biopsies from Cystic Fibrosis Patients

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

Modulation of Ca2+-activated Cl- secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia

Human airway epithelia express Ca-activated Cl channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K channels in CaCC-mediated Cl secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K channels, and by clotrimazole, an inhibitor of Ca-activated hSK4 K channels. The K channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca-activated Cl secretion in native human airway epithelia requires activation of Ca-dependent basolateral K channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease

Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated C1- secretion in human airway epithelia

Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)-dependent CI conductance. Activation of Cl secretion in airways depends on simultaneous activation of luminal CI channels and basolateral K channels. We determined the role of basolateral K conductance in cAMP-dependent Cl secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na conductance and defective cAMP-mediated Cl conductance. In non-CF tissues, Cl secretion was significantly inhibited by the chromanol 293B (10 μmol/liter), a specific inhibitor of K(V)LQT1 K channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 μmol/liter) simultaneously activated Cl and K conductance. The K conductance was reversibly inhibited by Ba and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K(V)LQT1. We conclude that the K channel K(V)LQT1 is important in maintaining cAMP-dependent Cl secretion in human airways. Activation of K(V)LQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl channel activity or alternative Cl channels could help to circumvent the secretory defect

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

<p>(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.</p

<i>CFTR</i> genotypes of CF patients with no detectable Cl⁻ secretion (CF_absent) and residual Cl⁻ secretion (CF_residual) in native rectal epithelia.

<p><i>CFTR</i> genotypes of CF patients with no detectable Cl⁻ secretion (CF_absent) and residual Cl⁻ secretion (CF_residual) in native rectal epithelia.</p

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

<p>(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. *<i>P</i><0.001, ^†<i>P</i><0.01 and ^¶<i>P</i><0.05.</p

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

<p>(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 <i>CFTR</i> 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. * <i>P</i><0.001 and ^†<i>P</i><0.01.</p

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.

<p>(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. *<i>P</i><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.</p