Pharmacology of CFTR Chloride Channel Activity. Physiol. Rev. 79, Suppl.: S109-S144, 1999. - The pharmacology of cystic fibrosis transmembrane conductance regulator (CFTR) is at an early stage of development. Here we attempt to review the status of those compounds that modulate the Cl- channel activity of CFTR. Three classes of compounds, the sulfonylureas, the disulfonic stilbenes, and the arylaminobenzoates, have been shown to directly interact with CFTR to cause channel blockade. Kinetic analysis has revealed the sulfonylureas and arylaminobenzoates interact with the open state of CFTR to cause blockade. Suggestive evidence indicates the disulfonic stilbenes act by a similar mechanism but only from the intracellular side of CFTR. Site-directed mutagenesis studies indicate the involvement of specific amino acid residues in the proposed transmembrane segment 6 for disulfonic stilbene blockade and segments 6 and 12 for arylaminobenzoate blockade. Unfortunately, these compounds (sulfonylureas, disulfonic stilbenes, arylaminobenzoate) also act at a number of other cellular sites that can indirectly alter the activity of CFTR or the transepithelial secretion of Cl-. The nonspecificity of these compounds has complicated the interpretation of results from cellular-based experiments. Compounds that increase the activity of CFTR include the alkylxanthines, phosphodiesterase inhibitors, phosphatase inhibitors, isoflavones and flavones, benzimidazolones, and psoralens. Channel activation can arise from the stimulation of the cAMP signal transduction cascade, the inhibition of inactivating enzymes (phosphodiesterases, phosphatases), as well as the direct binding to CFTR. However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.

The establishment of normal pediatric polysomnographic parameters is important for both clinical and research interests. Our objectives were to describe respiratory events, paradoxical breathing, periodic limb movements, and sleep architecture of children at the age of peak incidence of obstructive sleep apnea syndrome. We performed a retrospective cross-sectional analysis of a prospective cohort study of 66 children, 2-9 years old, at the Sleep Disorders Center at the Children's Hospital of Philadelphia. Subjects screened by questionnaire underwent a standard polysomnogram. The percent of total sleep time spent in sleep stages 1, 2, 3, 4, and rapid eye movement (REM) were 4 +/- 3%, 44 +/- 10%, 10 +/- 6%, 22 +/- 8%, and 21 +/- 6%, respectively. The arousal and awakening index was 11.2 +/- 4.3/hr. Respiratory events included a central apnea index of 0.08 +/- 0.14/hr, obstructive apnea index of 0.01 +/- 0.03/hr, and obstructive hypopnea index of 0.3 +/- 0.5/hr. The baseline arterial oxygen saturation (SpO2) was 97 +/- 1%, with a nadir of 92 +/- 3%. The index of periodic limb movements in sleep (PLMS) was 1.3 +/- 2.2/hr. Paradoxical breathing appeared significantly more frequent with piezo crystal effort belts (40 +/- 24% of epochs) than with respiratory inductive plethysmography (1.5 +/- 3% of epochs). We describe the occurrence of hypopneas during sleep, arousals and awakenings, and PLMS. We illustrate how different technologies can vary the apparent amount of paradoxical breathing. We also confirm previous data on the frequency distribution of sleep stages, SpO2, and relative rarity of respiratory events in this age group.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A- and ATP-regulated Cl- channel located in the apical membranes of epithelial cells. Previously Sheppard and Welsh (J. Gen. Physiol. 100: 573-591, 1992) showed that glibenclamide, a compound which binds to the sulfonylurea receptor and thus blocks nucleotide-dependent K+ channels, reduced CFTR whole cell current. The aim of this study was to identify the mechanism underlying this inhibition in cell-free membrane patches containing CFTR Cl- channels. Exposure to gliben-clamide caused a reversible reduction in current carried by CFTR which was paralleled by a decrease in channel open probability (Po). The decrease in Po was concentration dependent, and half-maximum inhibition (ki) occurred at 30 microM. Fluctuation analysis indicated a flickery-type block of open CFTR channels. Event duration analysis supported this notion by showing that the glibenclamide-induced decrease in Po was accompanied by interruptions of open bursts [i.e., an apparent reduction in the burst duration (Tburst)] with only a slight reduction in closed time (Tc). The plot of the corresponding open-to-closed (Tburst-1) and closed-to-open (Tc-1) rates as a function of glibenclamide concentration were consistent with a pseudo-first-order open-blocked mechanism and provided estimates of the on rate (kon = 1.17 microM-1S-1), the off rate (koff = 16 s-1), and the dissociation constant (Kd = 14 microM). The difference between the Ki (30 microM) and the Kd (14 microM) is the result expected for a closed-open-blocked model with an initial Po of 0.47. Since the initial Po was 0.50 +/- 0.02 (n = 12), we can conclude that glibenclamide blocks CFTR by a closed-open-blocked mechanism.

The low Ca 2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported [Yamauchi et al. Biochem Biophys Res Commun, 2005] that the epithelial Ca 2+ channels TRPV5 and TRPV6 are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca 2+ ] and transepithelial potential of the utricle (UP) were measured in Cl -/ exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a4 -/-mice exhibit reduced pH and UP and increased [Ca 2+ ]. Monolayers of primary cultures of rat semicircular canal duct (SCCD) cells were grown on permeable supports and cellular uptake of 45 Ca 2+ was measured individually from the apical and basolateral sides. Net uptake of 45 Ca 2+ was greater after incubation with 1,25(OH) 2 D 3 . Net 45 Ca 2+ absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [Ca 2+ ] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca 2+ absorption.