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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.

Studying membrane proteins at the molecular level represents a major challenge in biochemistry due to the complexity of the membrane in which they are embedded. As an important step towards a detailed understanding of their action and molecular functioning, current studies focus on membrane proteins reconstituted into artificial lipid environments. Such reconstituted systems allow for a more flexible choice of biochemical, biophysical, and microscopy techniques for characterizing the proteins. This review gives an overview of the methods currently available for reconstituting membrane proteins in a functional state into giant unilamellar vesicles, and discusses some key methods to verify successful reconstitution.

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)

Membrane transporters regulate multiple physiological processes including the pumping of ions, peptides and other metabolites such as neurotransmitters. Despite plethora of information available on transmembrane transporters, the possibility that transporters like all other enzymes studied to date display static and dynamic fluctuations in activity remains open. Importantly we currently ignore the impact such fluctuation have on the critical issue of pumping stoichiometry and therefore pumping efficiency. For example, several studies on P-type ATPases suggest substantially less amount of cations being transported to ATP hydrolyzed, possibly due to uncoupling of ATP-hydrolysis from cation transport during resting state.(1,2)Here, we present results from our ongoing investigation on time-resolved recording of single transporter events, using surface-based arrays of proteoliposomes.(3-5) Our single molecule studies have revealed fluctuations (on/off states) in the activity the P-type H+-ATPases-2 from Arabidopsis thaliana (AHA2). Insights from these on and off activity states for proton transporter will be used to estimate the true proton to ATP stoichiometry. Preliminary experiments were also performed to understand the role of the regulatory domain that is thought to be critical in coupling hydrolysis to proton coupling.(6)References:(1)Berman, M. C. Biochim. Biophys. Acta 2001, 1513, 95.(2)Venema, K. et al. J. Biol. Chem. 1995, 270, 19659.(3)Christensen, S. M. et al. Nat. Nanotechnol. 2012, 7, 51.(4)Hatzakis, N. S. et al. Nat. Chem. Biol. 2009, 5, 835.(5)Mathiasen, S. et. al. Nat. Methods 2014, 11, 931.(6)Pedersen, B. P. et al. Nature 2007, 450, 1111.

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.