Martin J. Hug

Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A

TMEM16A (anoctamin 1, Ano1), a member of a family of 10 homologous proteins, has been shown to form an essential component of Ca2+‐activated CU channels. TMEM16A‐null mice exhibit severe defects in epithelial transport along with tracheomalacia and death within 1 mo after birth. Despite its outstanding physiological significance, the mechanisms for activation of TMEM16A remain obscure. TMEM16A is activated on increase in intracellular Ca2+, but it is unclear whether Ca2+ binds directly to the channel or whether additional components are required. We demonstrate that TMEM16A is strictly membrane localized and requires cytoskeletal interactions to be fully activated. Despite the need for cytosolic ATP for full activation, phosphorylation by protein kinases is not required. In contrast, the Ca2+ binding protein calmodulin appears indispensable and interacts physically with TMEM16A. Openers of small‐ and intermediate‐conductance Ca2+‐activated potassium channels known to interact with calmodulin, such as 1‐EBIO, DCEBIO, or riluzole, also activated TMEM16A. These results reinforce the use of these compounds for activation of electrolyte secretion in diseases such as cystic fibrosis.—Tian, Y., Kongsuphol, P., Hug, M., Ousingsawat, J., Witzgall, R., Schreiber, R., Kunzelmann, K. Calmodulin‐dependent activation of the epithelial calcium‐dependent chloride channel TMEM16A. FASEB J. 25, 1058–1068 (2011). www.fasebj.org

Effect of ATP, carbachol and other agonists on intracellular calcium activity and membrane voltage of pancreatic ducts.

The pancreatic duct has been regarded as a typical cAMP-regulated epithelium, and our knowledge about its Ca2+ homeostasis is limited. Hence, we studied the regulation of intracellular calcium, [Ca2+]i, in perfused rat pancreatic ducts using the Ca2+-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, Vbl, of individual cells. The resting basal [Ca2+]i was relatively high, corresponding to 263±28 nmol/l, and it decreased rapidly to 106±28 nmol/l after removal of Ca2+ from the bathing medium (n=31). Carbachol increased [Ca2+]i in a concentration-dependent manner. At 10 μmol/l the fura-2 fluorescence ratio increased by 0.49±0.06 (n=24), corresponding to an increase in [Ca2+]i by 111±15 nmol/l (n=17). ATP, added to the basolateral side at 0.1 mmol/l and 1 mmol/l, increased the fluorescence ratio by 0.67±0.06 and 1.01±14 (n=46; 12), corresponding to a [Ca2+]i increase of 136±22 nmol/l and 294±73 nmol/l respectively (n= 15; 10). Microelectrode measurements showed that ATP (0.1 mmol/l) hyperpolarized Vbl from −62±3 mV to-70±3 mV, an effect which was in some cases only transient (n=7). This effect of ATP was different from that of carbachol, which depolarized Vbl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of Vbl (n=4). Several other putative agonists of pancreatic HCO3−secretion were also tested for their effects on [Ca2+]i. Bombesin (10 nmol/l) increased the fura-2 fluorescence ratio by 0.24±0.04 (n=8), neurotensin (10 nmol/l) by 0.25±0.04 (n=6), substance P (0.1 μmol/l) by 0.22±0.06 (n=6), and cholecystokinin (10 nmol/l) by 0.14±0.03 (n=7). Taken together, our studies show that Ca2+ homeostasis plays a role in pancreatic ducts. The most important finding is that carbachol and ATP markedly increase [Ca2+]i, but their different electrophysiological responses indicate that intracellular signalling pathways may differ.

THE ROLE OF EXOCYTOSIS IN THE ACTIVATION OF THE CHLORIDE CONDUCTANCE IN CHINESE HAMSTER OVARY CELLS (CHO) STABLY EXPRESSING CFTR

Abstract The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl–conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation ΔF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (Vm), the membrane conductance (Gm) and the membrane capacitance (Cm). Cm was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 μmol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on Vm and Gm in control CHO and ΔF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from –40 ± 1.5 to –32 ± 1.6 mV, n = 41) and enhanced Gm strongly from 5.0 ± 0.9 to 36 ± 3.9 nS (n = 65). The conductance increase was mostly for Cl–, because under stimulated conditions a reduction in bath Cl–concentration depolarized these cells further and significantly from –26 ± 1.8 to –10 ± 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br– > Cl– > I–(n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance Cm was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of ΔF508-CFTR in CHO cells induces a cAMP-activated Cl–conductance. The activation of this large conductance obviously proceeds with little if any exocytosis.

CHLORIDE CHANNELS IN THE LUMINAL MEMBRANE OF RAT PANCREATIC ACINI

Abstract Pancreatic acini secrete Na+, Cl–and H2O in response to secretagogues such as acetylcholine. Cl–channels in the luminal membrane are a prerequisite for this secretion. The properties of the corresponding conductance have previously been examined using whole-cell recordings. The present study attempts to examine the properties of the single channels in cell-attached and cell-free excised patches from the luminal membrane. To this end the pipettes were filled with an N-methyl-D-glucamine (NMDG+) chloride/gluconate solution. The voltage-clamp range was chosen to be pipette positive (cell negative, –60 to –130 mV) in order to increase the driving force for outward Cl–currents. Under resting conditions cell attached luminal patches had very few single-channel currents (12 out of 45 experiments). Their incidence was sharply increased by carbachol (CCH, 1 μmol/l) in 41 out of 45 experiments. The single-channel conductance of these channels was 1.97 ± 0.05 pS. The properties of these channels in excised patches were examined further: their single-channel conductance was 2.2 ± 0.07 pS (n = 59) and their conductance selectivity was I– > Br– > Cl– >> gluconate. None of the typical Cl–channel blockers (DIDS, NPPB, glibenclamide 100 μmol/l) blocked these channels. It is concluded that the luminal membrane of the rat pancreatic acinus possesses Cl–channels with very low conductance which are activated by carbachol.

The Na+2Cl–K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage

Abstract Effects of cAMP on Cl– secretion, intracellular Cl– activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl– secretion in a biphasic manner. The first and rapid phase corresponds to Cl– exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl– conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1–2 min) activation of the Na+2Cl–K+ cotransporter. During the first phase cytosolic Cl– activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl– activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl–K+ cotransport is transient cell shrinkage.

N-Acetyl-l-cysteine and its derivatives activate a Cl− conductance in epithelial cells

N-Acetyl-l-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (we) conductance (Gm) and voltage (Vm) of NAC and the congeners S-carboxymethyl-l-cysteine (CMC) andS-carbamyl-L-cysteine (CAC) andL-cysteine in normal and CF airway epithelial cells.L-Cysteine (1 mmol/1) had no detectable effect. The increase inGm (ΔGm) by the other compounds was concentration dependent and was (all substances at 1 mmol/1) 3.8 ± 1.4 nS (NAC; n = 11), 4.2 ± 1.0 nS (CMC;n = 16) and 3.8 ± 1.6 nS (CAC;n = 18), respectively. The changes in Gm were paralleled by an increased depolarization (ΔVm) when extracellular Cl− concentration was reduced to 34 mmol/1: under control conditions = -4.1 ± 2.1 versus 10.2 ± 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl− concentration was paralleled by a reduction ofGm by 2.1 ± 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl− conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/1). A Cl− conductance was also activated in HT29 colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPA1 cells, which do not express detectable levels of ΔF508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl− currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-l-methylxanthine (IBMX) (ΔGm = 0.08 ±0.04 μS;n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected cocytesGm was enhanced when intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (Gm = 4.5 ± 1.3 μS;n = 8).Gm was significantly increased by 0.74 ± 0.2 μS (n = 11) and 0.46 ± 0.1 μS (n = 10) when oocytes were exposed to NAC and CMC, respectively (both I mmol/1). In conclusion, NAC and its congeners activate Cl− conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.

Regulation of slowly activating potassium current (IKs) by secretin in rat pancreatic acinar cells

1 The secretagogue‐activated K+ conductance is indispensable for the electrogenic Cl− secretion in exocrine tissue. In this study, we investigated the effect of secretin and other cAMP‐mediated secretagogues on the slowly activating voltage‐dependent K+ current (IKs) of rat pancreatic acinar cells (RPAs) with the whole‐cell patch clamp technique. 2 Upon depolarization, RPAs showed IKs superimposed upon the instantaneous background outward current. Secretin (5 nm), vasoactive intestinal peptide (5 nm), forskolin (5 μm), isoprenaline (10 μm) or 3‐isobutyl‐1‐methylxanthine (IBMX, 0.1 mm) increased the amplitude of IKs two‐ to fourfold. 3 The physiological concentration of secretin (50 pm) had a relatively weak effect on IKs (160 % increase), which was significantly enhanced by transient co‐stimulation with carbachol (CCh) (10 μm). However, the secretin‐induced production of cAMP, which was measured by enzyme‐linked immunosorbent assay, was not augmented by co‐stimulation with CCh. 4 This study is the first to demonstrate the regulation of K+ channels in RPAs by cAMP‐mediated agonists. The IKs channel is a common target for both Ca2+ and cAMP agonists. The vagal stimulation under the physiological concentration of secretin facilitates IKs, which provides an additional driving force for Cl− secretion.

Calcium influx pathways in rat pancreatic ducts

A number of agonists increase intracellular Ca2+ activity, [Ca2+]i, in pancreatic ducts, but the influx/efflux pathways and intracellular Ca2+ stores in this epithelium are unknown. The aim of the present study was to characterise the Ca2+ influx pathways, especially their pH sensitivity, in native pancreatic ducts stimulated by ATP and carbachol, CCH. Under control conditions both agonists led to similar changes in [Ca2+]i. However, these Ca2+ transients, consisting of peak and plateau phases, showed different sensitivities to various experimental manoeuvres. In extracellular Ca2+-free solutions, the ATP-induced [Ca2+]i peak decreased by 25%, but the CCH-induced peak was unaffected; both plateaus were inhibited by 90%. Flufenamate inhibited the ATP-induced peak by 35%, but not the CCH-evoked peak; the plateaus were inhibited by 75-80%. La3+ inhibited the ATP-induced plateau fully, but that induced by CCH by 55%. In resting ducts, an increase in extracellular pH, pHe, by means of HEPES and HCO3/CO2 buffers, increased [Ca2+]i; a decrease in pHc had the opposite effect. In stimulated ducts the pH-evoked effects on Ca2+ influx were more pronounced and depended on the agonist used. At pHe 6.5 both ATP- and CCH-evoked plateaus were inhibited by about 50%. At pH 8.0 the ATP-stim-ulated plateau was inhibited by 27%, but that stimulated by CCH was increased by 72%. Taken together, we show that CCH stimulates Ca2+ release followed by Ca2+ influx that is moderately sensitive to flufenamate, La3+, depolarisation, it is inhibited by low pH, but stimulated by high pH. ATP stimulates Ca2+ release and probably an early Ca2+ influx, which is more markedly sensitive to flufenamate and La3+, and is both inhibited by low and high pH. Thus our study indicates that there are at least two separate Ca2+ influx pathways in pancreatic ducts cells.

Whole-cell conductive properties of rat pancreatic acini

Acetylcholine-controlled exocrine secretion by pancreatic acini has been explained by two hypotheses. One suggests that NaCl secretion occurs by secondary active secretion as has been originally described for the rectal gland of Squalus acanthias. The other is based on a “push-pull” model whereby Cl- is extruded luminally and sequentially taken up basolaterally. In the former model Cl- uptake is coupled to Na+ and basolateral K+ conductances play a crucial role, in the latter model, Na+ uptake supposedly occurs via basolateral non-selective cation channels. The present whole-cell patch-clamp studies were designed to further explore the conductive properties of rat pancreatic acini. Pilot studies in approximately 300 cells revealed that viable cells usually had a membrane voltage (Vm) more hyperpolarized than - 30 mV. In all further studies Vm had to meet this criterion. Under control conditions Vm was - 49 ± 1 mV (n = 149). The fractional K+ conductance (fK) was 0.13 ± 0.1 (n = 49). Carbachol (CCH, 0.5 μmol/1) depolarized to-19 ± 1.1 mV (n = 63) and increased the membrane conductance (Gm) by a factor of 2-3. In the seeming absence of Na+ [replacement by N-methyl-D-glucamine (NMDG+)] Vm hyperpolarized slowly to -59 ±2 mV (n = 90) and CCH still induced depolarizations to- 24 ± 2 mV (n = 34). The hyperpolarization induced by NMDG+ was accompanied by a fall in cytosolic pH by 0.4 units, and a very slow and slight increase in cytosolic Ca2+. (fK) increased to 0.34. The effect of NMDG+ on Vm was mimicked by the acidifying agents propionate and acetate (10mmol/l) added to the bath. The present study suggests that fK makes a substantial contribution to Gm under control conditions. The NMDG+ experiments indicate that the non-selective cation conductance contributes little to Vm in the presence of CCH. Hence the present data in rat pancreatic acinar cells do not support the push-pull model.

Expression of Cystic Fibrosis Transmembrane Conductance Regulator Alters the Responses to Hypotonic Cell Swelling and ATP of Chinese Hamster Ovary Cells

The aim of this study was to examine whether the stable expression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR) in Chinese hamster ovary (CHO) cells alters the properties of these cells towards hypotonic cell swelling and ATP. According to many previous studies this was not expected a priori, since overexpression of CFTR should not affect the conductive pathways upregulated by the purinergic agonist or cell swelling. Three types of CHO cells were examined: a control group of normal CHO cells; a group of CFTR-CHO cells stably expressing wild-type CFTR at high levels (CHO-CFTR), and a group ΔF508-CFTR-CHO cells, stably expressing the frequent mutation ΔF508 CFTR (CHO-ΔF508). Whole cell patch-clamp studies were performed to measure the membrane voltage (V_m), the membrane conductance (G_m), and the membrane capacitance (C_m). Hypotonic cell swelling (Hypo, 150 mosm/l) was used, because it activates Cl^– and K⁺ channels and enables the cell to extrude KCl in many cells, and ATP because it is known to activate Ca²⁺-regulated channels in a large variety of cells. Hypo depolarized all three types of cells. This depolarization was accompanied by an increase in Cl^– conductance. The selectivity of the conductance was I^– ≥ Br^– ≥ Cl^– in CHO cells, but Cl^– = Br^– = I^– in the CFTR cells. Even more surprising: ATP (100 µmol/l) hyperpolarized CHO and ΔF508 cells and predominantly enhanced K⁺ conductance, whilst it depolarized and increased mostly a Cl^– conductance in CFTR cells. The selectivity of this anion conductance was atypical for ATP: Br^– > Cl^– > I^–. C_m was increased by ATP and Hypo in all three types of cells. ATP enhanced cytosolic Ca²⁺ ([Ca²⁺]_i) in all three types of cells but did not enhance cAMP. These data indicate that the expression of CFTR profoundly alters the properties of CHO cells. Agonists which stimulate characteristic Ca²⁺-regulated channels now enhance a Cl^– conductance resembling the properties of CFTR-Cl^– conductance.