Evidence for ATP Interaction with Phosphatidylcholine Bilayers

Abstract

ATP is a fundamental intracellular molecule and is thought to diffuse freely through the cytosol. However, evidence obtained from nucleoside-sensing sarcolemmal ion channels and red blood cells suggest that ATP is compartmentalised or buffered, especially beneath the sarcolemma. Macromolecular crowding of the cytosolic compartment, tortuosity of cytoskeletal structures and non-hydrolysing binding sites on cytoskeletal proteins are some of the physical barriers proposed that could potentially reduce free diffusion of adenine nucleotides through the cytosol. In this study we compared ATP and adenosine using a spectroscopic study of RH421, a membrane bound electrochromic dye, impedance spectroscopy measurements with tethered phospholipid bilayers and differential scanning calorimetry to determine the interaction with zwitterionic phospholipid bilayers. We report a concentration dependent shift of the excitation spectrum of RH421 indicating an interaction between ATP and DOPC unilammelar vesicles, with little effect observed from adenosine. This was supported by a concentration dependent change in the calculated membrane conductance of tethered phospholipid bilayers with ATP and not adenosine. Concentration dependent alterations to the measured enthalpy of the DMPC main phase transition with ATP was also observed, which weren’t replicated with adenosine. These observations provide strong evidence for the interaction of ATP with zwitterionic phospholipid bilayers at physiologically relevant concentrations. An interaction between ATP and the phospholipid bilayer could create a population of ATP molecules with a divergent diffusion rate with respect to “free” ATP and thus establish spatially distinct areas of compartmentalised ATP beneath the sarcolemma.