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The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.

In eukaryotes, P-type adenosine triphosphatases (ATPases) generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. We monitored at the single-molecule level the activity of the prototypic proton-pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements, combined with a physical nonequilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 seconds) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters.

Regulated and selective transport of biomolecules across biological membranes is mediated by transporter proteins. These are essential in physiological processes ranging from electrical signaling in the nervous system to secretion of hormones and maintenance of electrochemical gradients. Though transporters are extensively studied, there are currently no techniques available to investigate their function at the single molecule level. We have developed a fluorescence-based assay to monitor thousands of individual nanoscale proteoliposomes in parallel by immobilizing them on functionalized glass surfaces (1-5).We recently extended this assay to allow real-time observation of substrate translocation mediated by single transporters reconstituted into liposomes. Here we present the development of the assay including characterization and optimization of the most critical system components including: minimization of leakage, in situ real-time calibration of absolute transport rates and increase of photostability for long term recordings (∼40min). We also present high-resolution single molecule activity recordings of the transporter protein, Arabidopsis thaliana H+-ATPase (AHA2).References:1. Mathiasen et al., Nature Methods. (2014) 11:931-342. Christensen. et al. Nat Nanotechnol. (2012) 7:51-53. Hatzakis. et al. Nat. Chem. Biol. (2009) 5:835-414. Bendix. et al. PNAS. (2009) 106:12341-65. Kunding. et al. Biophys J. (2008) 95:1176-88

Electrochemical gradients across cellular membranes control a plethora of vital biological processes. These gradients are generated by primary active transporters and are subsequently used to drive the exchange of other solutes through secondary active transporters and to facilitate signaling via ion channels. The macroscopic biological phenomena that channels and transporters give rise to are intimately connected to how they function at the single molecule level. For decades, patch clamp recording has been used to observe the functional dynamics of single ion channels revealing discrete on and off states, subconductance states, and other mechanistically important features that macroscopic experiments cannot probe(1). Currently, there aren’t any techniques available to investigate transporter function at the single molecule level, thus they are only studied using ensemble biochemical methods.For a decade we have been developing quantitative fluorescence microscopy based assays of arrayed proteoliposomes for investigating membrane proteins(2-6). Here we extended the platform to monitor the single-molecule activity and the regulation of a prototypic P-type ATPase, Arabidopsis thaliana H+-ATPase (AHA2). For the first time we have shown that individual proton pumps are not active continuously but rather transitioning between active and inactive states separated by a large activation barrier (kJMole−1). We found that the dynamics of these states form the basis of the regulation of the macroscopic activity either by regulatory R-domain, pH gradients, or ATP. Like for ion channels we often found that regulatory inputs do not affect the intrinsic pumping rates but rather active probabilities.References:1. Neher & Sajmann, Nature, (1976)2. Mathiasen et al., Nature Methods, (2014)3. Larsen et al., Nature Chemical Biology, (2015)4. Christensen et al., Nature Nanotechnology, (2012)5. Hatzakis et al., Nature Chemical Biology, (2009)6. Bendix et al., PNAS, (2009)

Previous studies have demonstrated an inverse relationship between plasma high density lipoproteins (HDL) cholesterol and coronary heart disease risk. In the present study we investigated prospectively the effect of a moderate physical conditioning programme on plasma lipids and lipoproteins, especially HDL-cholesterol and apolipoprotein A-I (apo A-I), the major apoprotein of HDL. Healthy, sedentary, middle-aged men were randomly selected and assigned either to a training group (n = 24, age 40 +/- 3.4, mean +/- SD) or to a control group (n = 13, age 39 +/- 5.0). Training consisted of various indoor and outdoor sports activities 45 min/day, 3 times/week for 12 weeks at an intensity of approximately 80% of measured maximal oxygen uptake (VO2 max). The trained subjects were studied at 4, 8 and 12 weeks. The training increased VO2 max by 12% (P less than 0.01). Increases were observed in both apo A-I (10%, P less than 0.02) and HDL-cholesterol (8%, P less than 0.02) after training, with significant increases already after 4 and 8 weeks, respectively. Furthermore, decreases in total plasma cholesterol (5%, P less than 0.004) and plasma triglycerides (26%, P less than 0.003) were found without changes in body weight, body composition, cigarette smoking, alcohol consumption or the percentage composition of dietary intake. Fasting serum-insulin concentrations decreased significantly during training. No changes were noted in the control group. The present study demonstrates prospectively that moderate physical training can increase HDL.