John P. Winpenny

Volume-activated chloride currents in pancreatic duct cells.

We have used the patch clamp technique to study volume-activated Cl− currents in the bicarbonatesecreting pancreatic duct cell. These currents could be elicited by a hypertonic pipette solution (osmotic gradient 20 mOsm/l), developed over about 8 min to a peak value of 91 ± 5.8 pA/pF at 60 mV (n = 123), and were inhibited by a hypertonic bath solution. The proportion of cells which developed currents increased from 15% in freshly isolated ducts to 93% if the ducts were cultured for 2 days. The currents were ATP-dependent, had an outwardly rectifying current/voltage (I-V) plot, and displayed time-dependent inactivation at depolarizing potentials. The anion selectivity sequence was: ClO4 = I = SCN > Br = NO3 > Cl > F > HCO3 > gluconate, and the currents were inhibited to a variable extent by DIDS, NPPB, dideoxyforskolin, tamoxifen, verapamil and quinine. Increasing the intracellular Ca2+ buffering capacity, or lowering the extracellular Ca2+ concentration, reduced the proportion of duct cells which developed currents. However, removal of extracellular Ca2+ once the currents had developed was without effect. Inhibiting protein kinase C (PKC) with either the pseudosubstrate PKC (19–36), calphostin C or staurosporine completely blocked development of the currents. We speculate that cell swelling causes Ca2+ influx which activates PKC which in turn either phosphorylates the Cl− channel or a regulatory protein leading to channel activation.

Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice

Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5±2.8 pA/pF (n=29) in wild-type cells, and about half that value, i.e. 12.4±1.6 pA/pF (n=20) in heterozygotes (P=0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527±162 pA/pF (n=24) from wild-type, 316±35 pA/pF (n=21) from heterozygote and 419±83 pA/pF (n=7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells.

Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2)

1 Functional characteristics and substrate specificity of the rat proton‐coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrate‐evoked current using the two‐electrode voltage‐clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2. 2 Amino (and imino) acid transport via rPAT2 was pH‐dependent, Na+‐independent and electrogenic. At extracellular pH 5.5 (in Na+‐free conditions) proline uptake was saturable (Km 172±41 μM), demonstrating that rPAT2 is, relative to PAT1, a high‐affinity transporter. 3 PAT2 preferred substrates are L‐α‐amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4‐ or 5‐membered heterocyclic amino and imino acids such as 2‐azetidine‐carboxylate, proline and cycloserine, where both D‐ and L‐enantiomers are transported. 4 The major restrictions on transport are side chain size (the ethyl group of α‐aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH2 groups, as in β‐alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O‐methyl esters show either reduced transport (alanine‐O‐methyl ester) or are excluded. 5 The structural characteristics that determine the substrate specificity of rPAT2 have been identified. This information should prove valuable in the design of selective substrates/inhibitors for PAT1 and PAT2.

Chloride channels and cystic fibrosis of the pancreas

Cystic fibrosis (CF) affects approximately 1 in 2000 people making it one of the commonest fatal, inherited diseases in the Caucasian population. CF is caused by mutations in a cyclic AMP-regulated chloride channel known as CFTR, which is found on the apical plasma membrane of many exocrine epithelial cells. In the CF pancreas, dysfunction of the CFTR reduces the secretory activity of the tubular duct cells, which leads to blockage of the ductal system and eventual fibrosis of the whole gland. One possible approach to treating the disease would be to activate an alternative chloride channel capable of bypassing defective CFTR. A strong candidate for this is a chloride channel regulated by intracellular calcium, which has recently been shown to protect the pancreas in transgenic CF mice. Pharmacological intervention directed at activating this calcium-activated Cl− conductance might provide a possible therapy to treat the problems of pancreatic dysfunction in CF.

Bestrophin expression and function in the human pancreatic duct cell line, CFPAC-1

Pancreatic duct epithelial cells (PDECs) have been shown to express calcium activated chloride channels (CaCCs) and there is evidence for their involvement in fluid secretion from these cells. The molecular identity of the CaCC in PDECs remains unknown. Recently, the bestrophin family of proteins have been proposed as a potential molecular candidate for CaCCs. Expression of bestrophins is strongly correlated with the function of CaCCs in a variety of tissues. In the present study, the expression of bestrophins has been investigated in the cystic fibrosis pancreatic duct cell line, CFPAC‐1. Iodide efflux analysis was used to characterise native CaCCs in CFPAC‐1 cell monolayers. Efflux was induced with the addition of UTP (100 μm, 10.2 ± 1.5 nmol min−1), which was blocked by the chloride channel blockers niflumic acid (81%) and DIDS (90%). The UTP‐stimulated iodide efflux was shown to be Ca2+ dependent and cAMP independent. RT‐PCR analysis of RNA isolated from CFPAC‐1 cells demonstrated positive identification of all four human bestrophin mRNAs. Western blot of CFPAC‐1 cell protein isolates with antibodies specific to human bestrophin 1 (hBest1) showed that hBest1 protein was expressed in this cell line. HBest1 was present on the cell surface, demonstrated using biotinylation and confocal imaging, as well as in the cytoplasm. SiRNA‐mediated silencing of hBest1 in CFPAC‐1 cells reduced the UTP‐stimulated iodide efflux by around 40%. This study provides evidence that the bestrophins are expressed in pancreatic duct cells and, more specifically, that hBest1 plays a role in the CaCCs found in these cells.

The CLCA Gene Family: Putative Therapeutic Target for Respiratory Diseases

The CLCA proteins were first shown to exist in bovine trachea and named as chloride channels calcium activated (CLCA) due to the calcium-dependent chloride conductance that appeared to be activated on expression of these proteins in trachea and other secretory epithelial cells. Since their initial discovery the CLCA gene family has grown extensively and family members have been identified in bovine, human, murine, equine and porcine tissues. The CLCA proteins appear to have a role to play in chloride conductance across epithelial cells and hence epithelial fluid secretion; cell-cell adhesion, apoptosis, cell cycle control and tumorgenesis and metastasis; mucous production and cell signalling in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). There are four human homologues; hCLCA1, hCLCA2, hCLCA3 and hCLCA4. Interest in these proteins has gathered pace with the description of hCLCA1s involvement in several human respiratory diseases. This review will describe the CLCA gene family and then move on to look at the growing body of evidence that suggests that at least hCLCA1 has an important role in the pathogenesis of respiratory disease such as asthma, COPD and cystic fibrosis (CF).

Volume-sensitive chloride currents in primary cultures of human fetal vas deferens epithelial cells

Using the patch-clamp technique, we have identified a large, outwardly rectifying, Cl−-selective whole-cell current in primary cultures of human vas deferens epithelial cells. Whole-cell currents were time- and voltage-dependent and displayed inactivation following depolarising pulses ≥ 60 mV. Currents were equally permeable to bromide (PBr/PCl = 1.05 ± 0.04), iodide (PI/PCl = 1.06 ± 0.07) and Cl−, but significantly less permeable to gluconate (PGluc/PCl = 0.23 ± 0.03). Currents spontaneously increased with time after establishing a whole-cell recording, but could be inhibited by exposure to a hypertonic bath solution which reduced inward currents by 68 ± 4%. Subsequent exposure of the cells to a hypotonic bath solution led to a 418 ± 110% increase in inward current, indicating that these currents are regulated by osmolarity. 4,4′-Diisothiocyanatostilbene-2,2′-disulphonic acid (100 μM) produced a rapid and reversible voltage-dependent block (60 ± 5% and 10 ± 7% inhibition of current, measured at ± 60 mV, respectively). Dideoxyforskolin (50 μM) also reduced the volume-sensitive Cl− current, but with a much slower time course, by 41 ± 13% and 32 ± 16% (measured at ± 60 mV, respectively). Tamoxifen (10 μM) had no effect on the whole-cell Cl− current. These results suggest that vas deferens epithelial cells possess a volume-sensitive Cl− conductance which has biophysical and pharmacological properties broadly similar to volume-sensitive Cl− currents previously described in a variety of cell types.

Properties and role of calcium-activated chloride channels in pancreatic duct cells

Publisher Summary This chapter discusses the properties and role of calcium-activated chloride channels in pancreatic duct cells. The exocrine pancreas is composed of two main cell types: acinar cells and duct cells. Acinar cells are specialized secretory cells responsible for the production and secretion of a variety of digestive enzymes plus a small amount of plasma-like isotonic fluid. The secretion of this NaCl – rich fluid involves the activation of calcium-regulated Cl – and cation channels. In the human pancreas, acinar cells represent approximately 85% by volume of the gland. The duct cells form the tubular structures that ramify throughout the gland as the branches of a tree. There are basically four types of ducts based on anatomical and histological criteria. The ductal cells are primarily involved in salt and fluid transport and provide a structural framework for the acini. Their main task is the regulated secretion of large volumes of a HCO 3 – -rich isotonic fluid. This secretion helps to solubilize and flush digestive enzymes secreted by acinar cells located at the ends of the smallest ducts, along the ductal tree and into the duodenum.

The anoctamin (TMEM16) gene family: calcium-activated chloride channels come of age.

This issue contains papers presented at the symposium entitled The anoctamin (TMEM16) gene family: calcium-activated chloride channels come of age, held on Wednesday 13 July, during the 2011 main meeting of the Physiological Society, University of Oxford, UK. The setting for the Symposium was the lecture theatre in the Le Gros Clark building, which is part of the Department of Physiology, Anatomy and Genetics. Chloride channels that are specifically regulated by changes in intracellular calcium, so-called calcium-activated chloride channels (CaCCs), perform diverse and important roles in a variety of tissues (Duran et al. 2010). These channels help to regulate olfaction and taste, neuronal and cardiac excitability, as well as fluid movement in different epithelial tissues throughout the body. Despite much information about the biophysical and functional aspects of CaCCs in many cell types, knowledge about the gene or genes that encode for these plasma membrane proteins has remained elusive. The recent discovery that the anoctamin 1 (ANO1)/TMEM16A gene encodes for the CaCC (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008) has heralded a huge breakthrough in our understanding of the molecular identity of this channel. This discovery has been the catalyst for a flurry of reports that have furthered our understanding of the physiological function of anoctamins in epithelial, sensory and muscle tissues. This symposium brought together leaders in the field of CaCC physiology. The symposium began with an elegant presentation by Luis Galietta (Istituto Giannina Gaslini, Italy), one of the three laboratories that first identified the ANO1 (TMEM16A) gene as encoding a CaCC, on the structural and functional relationships of ANO1 (TMEM16A) and ANO2 (TMEM16B; Scudieri et al. 2012). He described the different electrophysiological properties of the four main splice variants of ANO1 (a, b, c and d), which are derived by alternative splicing and exon skipping, as well as the fact that they are differentially expressed in various cell types. He also showed that ANO2 (TMEM16B) behaves as a CaCC, although the channel requires a higher intracellular calcium concentration and has faster activation and deactivation channel kinetics than ANO1. These structural and physiological differences of the splice variants and paralogues may eventually help to explain the differing properties reported for CaCCs in different cell types. One of the main unresolved questions of CaCC regulation is whether the protein is directly activated by calcium binding or whether it is regulated by an associated calcium-binding protein, such as calmodulin or calmodulin-dependent protein kinase II (CaMKII). Karl Kunzelmann (University of Regensburg, Germany) presented data for hANO1 expressed in HEK 293 cells, and showed that activation of the channel has an absolute requirement for ATP as well as being dependent on calmodulin, but not CaMKII (Kunzelmann et al. 2012). This is interesting, given that many tissues have CaCCs that appear to be dependent on CaMKII for activation. It may be that the splice variants of ANO1 underlie this difference. There are many questions regarding the function of the anoctamins in different tissues. Anna Menini (International School for Advanced Studies, Italy) gave a lovely presentation on the importance of CaCCs in olfaction (Pifferi et al. 2012). Odorant molecules bind to odoursensing receptors (OSRs), leading to an increase in calcium entry that activates CaCC current that amplifies the depolarization of the OSR cells. Anna presented immunohistochemical and electrophysiological evidence for ANO2 (TMEM16B) as the underlying chloride channel involved in this process. Interestingly, recent work from Jentsch’s group, using ANO2 knockout mice, supports ANO2 as the CaCC in the main olfactory epithelium; however, these mice seem to have no reduction in olfactory behavioural tasks (Billig et al. 2011). These data therefore question the

Effects of n-alkanols and a methyl ester on a transient potassium (IA) current in identified neurones from Helix aspersa

1. A two‐microelectrode voltage clamp was used to determine the effects of n‐butanol, n‐hexanol, n‐octanol, n‐decanol and methyl hexanoate on a transient potassium (IA) current in identified Helix aspersa neurones. Experiments were carried out at a temperature of 10‐12 degrees C. 2. Each n‐alkanol reversibly reduced the amplitude of the IA current. Logarithmic dose‐response curves for the current reduction by each homologue were sigmoidal and had slope factors of around four. The concentrations required to reduce the peak (with time) current at ‐30 mV by 50% (ED50 +/‐ fitted standard error) were: 57 +/‐ 5 mM (n‐butanol); 2.0 +/‐ 0.1 mM (n‐hexanol); 0.28 +/‐ 0.02 mM (n‐octanol) and 0.016 +/‐ 0.001 mM (n‐decanol). Methyl hexanoate also reduced the current amplitude, with an ED50 of 1‐2 mM. The Helix IA current thus showed a similar sensitivity to n‐alkanols to that of squid and rat sodium currents but was rather more sensitive than the squid delayed rectifier potassium current. 3. The n‐alkanol ED50 concentrations were used to calculate a standard free energy per methylene group for adsorption to a site of action in the cell of ‐3.1 +/‐ 0.2 kJ/mol. This suggested a hydrophobic site or sites of action. The regularity of the change in free energy with chain length was maintained up to, and including, n‐decanol. This implied that the site(s) could accommodate a ten‐carbon chain as readily as an eight‐carbon chain. 4. The voltage dependencies of IA current activation and steady‐state inactivation were not consistently altered by treatment with n‐alkanols at concentrations around or above their current suppression ED50 concentrations. 5. The kinetics of current activation and inactivation were affected, particularly by lower chain length compounds. At 60 mM n‐butanol reduced the time constant for development of inactivation of open channels (tau b) by 56%, while 0.016 mM n‐decanol produced only a 13% reduction. n‐Butanol (60 mM) also caused a substantial (76%) reduction in the time constant for development of inactivation in channels which were presumed to be closed. The effects of n‐alkanols on the current time‐to‐peak (tc) were complex, showing both increases and decreases, but these actions also declined with chain length. Methyl hexanoate (1 mM) reduced tau b by around 30% and tc by around 20%. 6. n‐Alkanols have now been shown to inhibit a number of voltage‐gated ion conductances.(ABSTRACT TRUNCATED AT 400 WORDS)