Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

In droplet microfluidics, aqueous droplets are typically separated by an oil phase to ensure containment of molecules in individual droplets of nano-to-picoliter volume. An interesting variation of this method involves bringing two phospholipid-coated droplets into contact to form a lipid bilayer in-between the droplets. These interdroplet bilayers, created by manual pipetting of microliter droplets, have proved advantageous for the study of membrane transport phenomena, including ion channel electrophysiology. In this study, we adapted the droplet microfluidics methodology to achieve automated formation of interdroplet lipid bilayer arrays. We developed a ‘millifluidic’ chip for microliter droplet generation and droplet packing, which is cast from a 3D-printed mould. Droplets of 0.7–6.0 μL volume were packed as homogeneous or heterogeneous linear arrays of 2–9 droplets that were stable for at least six hours. The interdroplet bilayers had an area of up to 0.56 mm2, or an equivalent diameter of up to 850 μm, as determined from capacitance measurements. We observed osmotic water transfer over the bilayers as well as sequential bilayer lysis by the pore-forming toxin melittin. These millifluidic interdroplet bilayer arrays combine the ease of electrical and optical access of manually pipetted microdroplets with the automation and reproducibility of microfluidic technologies. Moreover, the 3D-printing based fabrication strategy enables the rapid implementation of alternative channel geometries, e.g. branched arrays, with a design-to-device time of just 24–48 hours

Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed "induced fit," which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolution of these positions, and because in crystal structures of ABC domains, interactions between these positions appear to be modulated by ATP binding. We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Our results suggest state-dependent changes in coupling between two of the three positions (1296 and 1303) and are consistent with a model that assumes a toggle switch-like interaction pattern during the intra-NBD2 induced fit in response to ATP binding. Stabilizing interactions of F1296 and N1303 present before ATP binding are replaced by a single F1296-N1303 contact in ATP-bound states, with similar interaction partner toggling occurring during the much rarer ATP-independent spontaneous openings

Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network

Regulation of CFTR and its contribution to other epithelial Cl- channels

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a cAMP dependent Cl- channel that is expressed mainly on the apical membrane of epithelial cells. CFTR plays an essential role in electrolyte and water homeostasis. Defects in CFTR cause CF disease, which is the most common lethal genetic disorder among Caucasians. Besides functioning as a Cl- channel, CFTR also acts as a regulator of other channels and transporters. Although CFTR has been known for more than 20 years, many questions regarding CFTR regulations and functions remain to be answered. In the present thesis, we performed various experiments in an attempt to understand CFTR in several aspects, including CFTR-regulation by AMPK in vitro and in vivo, the role of the S573C CFTR mutation for the risk to develop pancreatitis, and a novel role of CFTR in proton sensing and regulating CaCC. Regulation of CFTR by AMPK in vivo The present study has clarified the role of AMPK for epithelial Cl- transport in vivo using AMPKα1-/- mice. The data demonstrate a significant role of AMPK in regulating CFTR. In real short circuit Ussing chamber experiment, an increase in CFTR Cl- current is observed in the colon of AMPKα1-/- mice when exposed to cAMP. Unlike wild type littermates, the elevated cAMP-dependent Cl- current in the AMPKα1-/- colon is insensitive to the AMPK activator phenformin. Furthermore, rectal potential difference (RPD) measurements indicate that AMPKα1-/- mice have an increased RPD both non-stimulated and after exposure to cAMP. These data suggested enhanced activity of CFTR in the large intestine of AMPKα1-/- mice. In correlation to previous in vitro observations, data from the present study demonstrate that AMPK inhibits CFTR in vivo. Mechanistic insight into control of CFTR by AMPK To identify the AMPK phosphorylation sites in CFTR and to find the mechanisms underlying AMPK regulation of CFTR, Xenopus oocytes were used to overexpress different CFTR mutations, and the whole cell currents of the overexpressed oocytes were measured using the double electrode voltage clamp (DEVC) technique. In contrast to previous observations, this study demonstrates that S737 and S768 are phosphorylated by AMPK rather than PKA. In Xenopus oocytes, single or multiple mutations of S737 and S768 to alanine increase CFTR whole cell currents around 4-fold. Moreover, in non-activated cells, S737A and S768A increase the basal CFTR Cl- conductance around 5-fold. The elevated conductance is insensitive to the AMPK inhibitor phenformin and the AMPK activator compound C. Taken together, we demonstrate that AMPK inhibits PKA activation of CFTR. Moreover, AMPK indeed constitutively phosphorylates CFTR and keeps the channel shut at basal levels. We associated this finding to a spatiotemporal regulation of CFTR by cAMP. Data from the present study suggest a constitutive inhibition of CFTR by a tonic baseline AMPK activity. Because AMPK can also be activated without any detectable changes in the global AMP/ATP ratio, it is likely that AMPK regulation of CFTR is also controlled locally. The scenario can perhaps be described as AMP being generated by a Shank2/PDE complex bound to CFTR’s PDZ domain. PDE degrades cAMP to AMP, increasing the AMP/ATP ratio close to CFTR, and thus activating AMPK. The activated AMPK therefore inhibits CFTR and keeps the channel close under non-stimulated conditions. Metformin treatment of diabetes mellitus increases the risk of pancreatitis in patients bearing the CFTR-mutation S573C Metformin is an AMPK activator and is a drug used for the treatment of diabetes type II. Lactic acidosis is a common secondary complication of metformin therapy, especially in patients with renal dysfunction, alcohol abuse, or liver disease. Patients with renal failure have been reported to develop the pancreatitis after metformin therapy. We examined the effects of metformin treatment on a pancreatitis-related S573C-CFTR mutation. The results demonstrate a slight but significant reduction of S573C-CFTR whole cell currents that are further inhibited by metformin. On the other hand, metformin treatment of wtCFTR does not affect whole cell current induced by cAMP. These data suggest that the S573C mutation increases the sensitivity towards AMPK, However in vitro phosphorylation experiments reveal the same pattern of AMPK phosphorylation in wt and S573A-CFTR. Because lactic acidosis has been found during metformin therapy, and because CFTR is essential to control the pH balance in the pancreas, we challenged wt and S573C CFTR expressing oocytes by exposing them to acidic pH. The results demonstrate that intracellular acidificaiton reduces activation of wtCFTR, but almost completely abolishes Cl- currents produced by S573C-CFTR. Taken together these data imply that patients carrying the S573C mutation have only a slight defect in CFTR Cl- currents. However, under metformin treatment, the Cl- transport in patients carrying the S573C mutation is largely reduced. Thus S573C-carrier have a higher risk in developing a pancreatitis after metformin therapy. CFTR induces acid sensing and H+ activated Cl- transport In the last section of the present thesis we identified a novel role of CFTR for detection of the extracellular proton concentration and regulation of the Ca2+ activated Cl- current. In wtCFTR expressing Xenopus oocytes, we observed a Ca2+ activated Cl- current after exposure of the oocytes to extracellular acidic pH. This current was strongly outwardly rectifying, sensitive to DIDS and NPPB, and required intracellular Ca2+ for the activation. We hypothesize that CFTR translocate H+-receptors to the plasma membrane and extracellular protons binding to these receptors induces an increase in intracellular [Ca2+]. We speculate that CFTR-dependent H+-sensing may be essential for bone metabolism, since CFTR is expressed in bone cells and because CF-patients suffer from osteoporosis

Regulation of Cl− secretion by AMPK in vivo

Previous in vitro studies suggested that Cl− currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the α1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport–metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl− secretion by AMPK in vivo. Using AMPKα1−/− mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl− secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKα1−/− and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl− conductance in basolaterally permeabilized colonic epithelium from AMPKα1+/+ but not AMPKα1−/− mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl− secretion in epithelial tissues of AMPKα1−/− mice, but not in wild-type littermates. There was no effect on Ca2+-mediated Cl− secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl− secretion was enhanced in the colon of AMPKα1−/− mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl− secretion mediated by CFTR is controlled by AMPK in vivo

Microfluidic electrochemical multiplex detection of bladder cancer DNA markers

Bladder cancer (BC) is one of the most difficult cancers to diagnose, and is reportedly the most expensive cancer to treat and monitor due to the lack of accurate chemical sensors; only limited methods are available, which usually are restricted to cytology and cystoscopy. Thus a system is urgently needed to detect biomarkers that give an unambiguous diagnosis on BC, as it currently is among the top five cancers occurring. Here we present a multi-sensor microchip array which efficiently detects three specific bladder cancer DNA markers simultaneously with a detection limit of 250 fM, which is well below the amount of DNA markers found in urine samples. The detection is based on the electrochemical response of a free-base porphyrin marker which is embedded in a molecular beacon; this system can easily be adapted to detect other DNA markers and developed for field applications, including point-of-care diagnostics, and gives an unambiguous readout within 20 minutes

Kunzelmann K. Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A

-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.or