Calcium-activated Cl(-) Channels Research Articles

Sesquiterpene lactones improve secretory diarrhea symptoms by inhibiting intestinal Ca2+-activated Cl– channel activities directly and indirectly

Secretory diarrhea caused by bacteria and viruses is usually accompanied by activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl– channels (CaCCs) in the intestinal epithelium. Inhibition of CFTR and CaCCs activities significantly reduces fluid losses and intestinal motility in diarrheal diseases. For this reason, CFTR and CaCCs are potential targets of therapeutic drug screening. Here, we reported that the sesquiterpene lactones, alantolactone (AL) and isoalantolactone (iAL), significantly inhibited ATP and Eact-induced short-circuit currents in T84, HT-29 and Fischer rat thyroid (FRT) cells expressing transmembrane protein 16A (TMEM16A) in a concentration-dependent manner. AL and iAL also inhibited the CaCC-mediated short-circuit currents induced by carbachol in the mouse colons. Both compounds inhibited forskolin-induced currents in T84 cells but did not significantly affect mouse colons. In vivo studies indicated that AL and iAL attenuated gastrointestinal motility and decreased watery diarrhea in rotavirus-infected neonatal mice. Preliminary mechanism studies showed that AL and iAL inhibited CaCCs at least partially by inhibiting Ca2+ release and basolateral membrane K+ channels activity. These findings suggest a new pharmacological activity of sesquiterpene lactone compounds that might lead to the development of treatments for rotaviral secretory diarrhea.

Unravelling the requirement for host cell chloride channels during HRSV infection

Ion channels are a diverse class of transmembrane proteins that selectively allow ions across membranes, influencing a multitude of cellular processes. Modulation of these channels by viruses is emerging as an important host-pathogen interaction that regulates critical stages of the virus multiplication cycle including entry, replication and egress. Human respiratory syncytial virus (HRSV) causes severe respiratory tract infections globally and is one of the most lethal respiratory pathogens for infants in developing countries, with frequent development of bronchiolitis. Furthermore, it is the most significant cause of hospitalisation of infants in the UK. Evidence also indicates that severe childhood HRSV infection contributes towards the increased incidence of adult asthma. No HRSV vaccine is available, and currently the only treatment is immunoprophylaxis which is prohibitively expensive and only moderately effective; thus new treatment options are required. Utilising GFP-expressing HRSV in combination with an extensive panel of channel specific pharmacological inhibitors, we have identified an important role of cellular chloride (Cl-) channels during HRSV infection. Interestingly, pharmacological inhibition of specific Cl- channel families has ruled out involvement of the CFTR and instead highlighted a critical requirement for calcium-activated Cl- channels (CaCCs). Time of addition studies using CaCC blockers have indicated that these channels play a post-entry role during HRSV infection. Using genetic knockdown techniques we have isolated a single channel of interest and are now further investigating its role in facilitating HRSV multiplication, as well as assessing the importance of Cl- channels in replication cycles of other negative sense RNA viruses.

Lineage-Specific Expression of Bestrophin-2 and Bestrophin-4 in Human Intestinal Epithelial Cells

Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3- or Cl-. Bestrophin genes represent a newly identified group of calcium-activated Cl- channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.

Chloride channels in stroke

Vascular remodeling of cerebral arterioles, including proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs), is the major cause of changes in the cross-sectional area and diameter of the arteries and sudden interruption of blood flow or hemorrhage in the brain, ie, stroke. Accumulating evidence strongly supports an important role for chloride (Cl(-)) channels in vascular remodeling and stroke. At least three Cl(-) channel genes are expressed in VSMCs: 1) the TMEM16A (or Ano1), which may encode the calcium-activated Cl(-) channels (CACCs); 2) the CLC-3 Cl(-) channel and Cl(-)/H(+) antiporter, which is closely related to the volume-regulated Cl(-) channels (VRCCs); and 3) the cystic fibrosis transmembrane conductance regulator (CFTR), which encodes the PKA- and PKC-activated Cl(-) channels. Activation of the CACCs by agonist-induced increase in intracellular Ca(2+) causes membrane depolarization, vasoconstriction, and inhibition of VSMC proliferation. Activation of VRCCs by cell volume increase or membrane stretch promotes the production of reactive oxygen species, induces proliferation and inhibits apoptosis of VSMCs. Activation of CFTR inhibits oxidative stress and may prevent the development of hypertension. In addition, Cl(-) current mediated by gamma-aminobutyric acid (GABA) receptor has also been implicated a role in ischemic neuron death. This review focuses on the functional roles of Cl(-) channels in the development of stroke and provides a perspective on the future directions for research and the potential to develop Cl(-) channels as new targets for the prevention and treatment of stroke.

The development of Crofelemer: Connecting ethnobotany, conservation, biocultural diversity, global public health and indigenous knowledge

Crofelemer is a novel first in class compound extracted, isolated and purified from the stem bark latex of the widespread pioneer tree species Croton lechleri, (Sangre de Drago). The natural distribution of this species includes the Northwest Amazon regions of Colombia, Ecuador, Peru, Bolivia and adjacent regions of the Brazil. The latex of this species has been utilized by numerous indigenous peoples of the western Amazon basin orally for the treatment of diarrhea, topically for wound healing and many other therapeutic applications. Crofelemer is an acid-labile oligomeric proanthocyanidin. The primary monomers include (+)-gallocatechin; (-)-epigallocatechin; (+)-catechin and (-)-epicatechin. The oligomer predominantly contains 7–11 linked monomers in varying ratios and an average molecular weight ranges from 1800 to 2200 Da. A new drug application (NDA) for crofelemer for the treatment of HIV/AIDS related chronic diarrhea was submitted on December 5, 2011 to the US FDA and was granted a 6 month priority review designation. Crofelemer's antidiarrheal activity is mediated primarily through its action on two chloride channels. Specifically the compound reduces fluid loss and Cl- secretion through inhibition of both the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium activated Cl- channels (CaCC) in the intestinal epithelium. The drug has minimal systemic absorption and has demonstrated an excellent safety profile in global clinical trials with >2,000 patients dosed across multiple trials conducted in numerous countries. Most recently crofelemer demonstrated excellent efficacy and safety in a pivotal Phase 3 trial in HIV patients (ADVENT) in reducing secretory diarrhea with a p-value of 0.0096. Clinical trials have also been conducted at the International Center for Diarrheal Disease Research (ICDDR) in Dhaka, Bangladesh, demonstrating significant reduction in fluid loss in patients with cholera-induced diarrhea, a life-threatening condition that most recently killed 7,000 people Haiti. One goal of Napo is to make certain that crofelemer can be made available to help save lives in cholera outbreaks wherever they may occur in the world.

Translational Approaches for Pharmacotherapy Development for Acute Diarrhea

Areport is presented of a National Institute of Diabetes and Digestive and Kidney Diseases–sponsored single topic conference designed to determine scientific advances needed to encourage development of pharmacotherapy for diarrheal diseases. Acute diarrheal diseases are a global public health problem in developing and developed countries. They are a leading cause of mortality and morbidity for children under the age of 5 worldwide and have a major economic impact in developed countries. Although oral rehydration solution (ORS) along with oral zinc therapy has reduced mortality owing to acute diarrheal diseases, there are essentially no other approved, safe, and effective drugs to decrease stool volume and prevent fluid loss. On September 25–27, 2011, the National Institute of Diabetes and Digestive and Kidney Diseases convened a workshop on Translational Approaches for Pharmacotherapy Development for Acute Diarrhea to assess current treatments for acute diarrhea, to identify common host pathways targeted by infectious agents associated with acute diarrhea, to evaluate which pathophysiologic mechanisms are most clinically relevant as potential targets, and to identify promising areas for translational research.

The Role of Capsaicin in Spontaneous Pacemaking Activity in Gastrointestinal Tract

Interstitial cells of Cajal (ICCs) are the pacemaking cells in the gastrointestinal (GI) tracts that generate the rhythmic oscillations in the membrane potential which were known as slow waves recorded in native smooth muscle tissue preparations in the past. Auto-rhythmicity in GI tracts is an exclusive property of ICCs and the spread of slow waves through GI tracts occurs electrically, and not by the active regeneration properties of ICCs and smooth muscles. Pacemaking ICCs are organized into electrically coupled networks within discrete areas of the stomach, small intestine, and colon.1,2 ICCs were originally identified by Santiago Ramon y Cajal and were characterized by morphological criteria until the discovery that these cells express the receptor tyrosine kinase Kit.3 Subsequent studies determined that Kit immunoreactivity in the muscularis propria of the GI tract is restricted to 2 cell types: mast cells4 and ICCs. Kit-positive ICCs are distributed throughout the GI tract as well as in other smooth muscle tissues.5 All regions of the GI tract contain ICCs but the location within the muscularis propria varies according to region or species.6 In the myenteric plexus region, ICCs form a network between the muscle layers forming a mesh around the ganglia and are considered as major pacemaker cells. A network of deep muscular plexus ICCs is present in the small intestine between the inner and outer circular muscle layers.6 In the colon and parts of the gastric antrum, submuscular ICCs are located outside the circular muscle layer.7 Intramuscular ICCs are distributed through the longitudinal and circular muscle layers and are considered to be involved in neurotransmission from nerve terminals. Septal ICCs are found between the fascicles of muscle in humans and other large species. These can be considered as one type of intramuscular ICCs.8 Stellate, subserosal ICCs are observed on the boundary between the longitudinal muscle and the serosa in the colon of mice.9 Capsaicin (8-methyl-N-vanillyl-6-ninenamide) activates afferent fiber endings and capsaicin sensitive primary afferent neurons participate in the regulation of GI motility.10 Furthermore, capsaicin induces contraction in the guinea-pig ileum11 and relaxation in human small and large intestines.10 Namely, it is known that capsaicin may have excitatory action on animal GI motility but inhibitory on human. However, information about the effect of capsaicin on human GI motility is somewhat controversial. Some reports showed the oral administration of capsaicin to fail in showing a significant effect on GI motility in human12-14 but there are reports that capsaicin could contribute to protection of the esophagus and could promote retention of the irritant in the stomach and faster transit through the small bowel.15 However, there are no reports on the direct effect of the capsaicin on ICCs that have important roles for regulation of GI motility as pacemaker cells. In this issue of the Journal of Neurogastroenterology and Motility, Choi et al examined the role of capsaicin on ICC in mouse small intestine. They showed that capsaicin inhibited the pacemaker activity of ICC dose-dependently. The effect of capsaicin, however, is not through the transient receptor potential of the vanilloid type 1 (TRPV1) channel because capsazepine did not block the effect of capsaicin. Consistently with this result, they also showed in their previous study that TRPV1-immunopositive cells were not co-localized with c-Kit positive tissue layer.16 The effect of capsaicin was not through nitric oxide release from ICCs, because there was no influence on capsaicin-induced action by L-NAME, an inhibitor of nitric oxide synthase in their study. Choi et al. proposed that further studies are needed to investigate the direct effect of capsaicin on pacemaker ion channels like TRPC4 or TRPM7 responsible for pacemaking activity in ICCs.17 Interestingly, when the action of capsaicin was examined in the intracellular calcium oscillation, capsaicin completely abolished the calcium oscillation in ICCs. Recently, 3 research groups showed simultaneously the calcium activated Cl channels to be required for pacemaking activity using TMEM16A knockout mice.18-20 Capsaicin might inhibit pacemaking activity of ICCs through the decreased activity of calcium activated Cl channels due to completely abolished calcium oscillations by application of capsaicin to ICCs. The effect of capsaicin seems more complicated because capsaicin acts on ICCs as well as afferent nerve endings of GI tract. In the future, more sophisticated studies are needed to clarify the role of capsaicin in GI tract considering ICCs and species differences.

Exposure to cigarette smoke condensate reduces calcium activated chloride channel transport in primary sinonasal epithelial cultures

The cystic fibrosis transmembrane conductance regulator (CFTR) serves as a predominant Cl(-) transport conduit in airway epithelium and is inhibited by cigarette smoke in vitro and in vivo. Activation of secondary Cl(-) transport pathways through calcium-activated Cl(-) channels (CaCC) has been postulated as a mechanism to bypass defects in CFTR-mediated transport. Because it is not known whether CaCCs are also inhibited by tobacco exposure, the current study was designed to investigate the effect of cigarette smoke condensate (CSC) on CaCC transport. In vitro study. Well-characterized primary murine nasal septal epithelial (MNSE) and human sinonasal epithelial (HSNE) cultures were exposed to CSC in Ussing chambers. We monitored CaCC short-circuit current through stimulation of P2Y purinergic receptors with uridine triphosphate or adenosine triphosphate and selective inhibition of the CFTR-dependent secretory pathway. Characterization of CaCC current was also accomplished in primary airway cells derived from transgenic CFTR(-/-) (knockout) murine models. Change in CaCC-mediated current (DeltaI(SC) representing transepithelial Ca-mediated Cl(-) secretion in muA/cm(2)) was significantly decreased in CSC-exposed wild type MNSE when compared to controls (32.8 +/- 4.6 vs. 47.5 +/- 2.3; respectively; P < .02). A similar effect was demonstrated in CFTR(-/-) MNSE cultures (33.4 +/- 2.8 vs. 38.6 +/- 2.0; P < .05>. HSNE cultures also had a significant reduction in I(SC) (16.1 +/- 0.6 vs. 22.7 +/- 0; P = .008). CSC affects multiple pathways fundamental to airway ion transport, including both cyclic adenosine monophosphate and calcium activated Cl(-) transport. Inhibition of Cl(-) transport may contribute to common diseases of the airways, such as chronic rhinosinusitis and chronic obstructive pulmonary disease.

Inhibition of vascular calcium‐gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl(-) channels (CaCCs) and large conductance, calcium-gated K(+) channels (K(Ca)1.1). The goal of the present study was to ascertain whether blockers of K(Ca)1.1 inhibited calcium-activated Cl(-) currents (I(ClCa)) and if the pharmacological overlap between K(Ca)1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K(+) channels. Whole-cell Cl(-) and K(+) currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca(2+)]. The selective K(Ca)1.1 blocker paxilline (1 microM) inhibited I(ClCa) by approximately 90%, whereas penitrem A (1 microM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I(ClCa) by approximately 20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca(2+)-binding domain of murine K(Ca)1.1 had no effect on I(ClCa) while inhibiting spontaneous K(Ca)1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K(+) channels (K(Ca)2.1 and K(Ca)2.3), namely 1-EBIO, (100 microM); NS309, (1 microM); TRAM-34, (10 microM); UCL 1684, (1 microM) had no effect on I(ClCa). These data show that the selective K(Ca)1.1 blockers also reduce I(ClCa) considerably. However, the pharmacological overlap that exists between CaCCs and K(Ca)1.1 does not extend to the calcium-binding domain or to other calcium-gated K(+) channels.

Transmembrane Protein 16A (TMEM16A) Is a Ca2+-regulated Cl– Secretory Channel in Mouse Airways

For almost two decades, it has been postulated that calcium-activated Cl(-) channels (CaCCs) play a role in airway epithelial Cl(-) secretion, but until recently, the molecular identity of the airway CaCC(s) was unknown. Recent studies have unequivocally identified TMEM16A as a glandular epithelial CaCC. We have studied the airway bioelectrics of neonatal mice homozygous for a null allele of Tmem16a (Tmem16a(-/-)) to investigate the role of this channel in Cl(-) secretion in airway surface epithelium. When compared with wild-type tracheas, the Tmem16a(-/-) tracheas exhibited a >60% reduction in purinoceptor (UTP)-regulated CaCC activity. Other members of the Tmem16 gene family, including Tmem16f and Tmem16k, were also detected by reverse transcription-PCR in neonatal tracheal epithelium, suggesting that other family members could be considered as contributing to the small residual UTP response. TMEM16A, however, appeared to contribute little to unstimulated Cl(-) secretion, whereas studies with cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice and wild-type littermates revealed that unstimulated Cl(-) secretion reflected approximately 50% CFTR activity and approximately 50% non-Tmem16a activity. Interestingly, the tracheas of both the Tmem16a(-/-) and the CFTR(-/-) mice exhibited similar congenital cartilaginous defects that may reflect a common Cl(-) secretory defect mediated by the molecularly distinct Cl(-) channels. Importantly, the residual CaCC activity in Tmem16a(-/-) mice appeared inadequate for normal airway hydration because Tmem16a(-/-) tracheas exhibited significant, neonatal, lumenal mucus accumulation. Our data suggest that TMEM16A CaCC-mediated Cl(-) secretion appears to be necessary for normal airway surface liquid homeostasis.