Extravesicular chloride ion regulates the uptake of ATP, sulfate, and inorganic phosphate by membrane vesicles from bovine adrenal chromaffin granules

ATP stored in secretory vesicles is released into the extracellular space through exocytosis, acting as an intercellular messenger in purinergic signaling. The vesicular nucleotide transporter (VNUT) is crucial for filling the vesicles with ATP. However, the mechanisms that regulate this transport are not yet fully understood. This study explores how anions influence ATP uptake in membrane vesicles from the bovine adrenal chromaffin granules. Our findings indicate that ATP uptake is driven by the membrane potential established by the vacuolar H⁺-ATPase and is sensitive to diisothiocyanostilbene disulfonic acid, phosphoenolpyruvate, atractyloside, and 2'(or-3')-O-(N-Methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP). Notably, extravesicular Cl^- significantly influences the ATP uptake: facilitation of ATP uptake occurs at millimolar Cl^- concentrations, peaking between 5 to 20 mM, although it declines sharply at higher Cl^- levels. A considerable amount of ATP is taken up by nigericin plus K⁺ without Cl^-. The membrane vesicles took up radiolabeled Cl^- in an ATP- and membrane potential-dependent fashion, which is partially sensitive to 5-nitro-2-(3-phenylpropylamino)benzoic acid and MANT-ATP but not to AMP. Sulfate and inorganic phosphate (Pi) at mM levels inhibit ATP uptake and are taken up in an ATP-dependent manner, exhibiting similar energetics, Cl^- dependency, and pharmacological profile to ATP uptake. In the mouse adrenal gland membranes, the uptake of sulfate and Pi depended on ATP, while this is not the case for those from VNUT knockout mice. These results suggest that VNUT transports not only nucleotides but also inorganic oxyanions, utilizing Cl^- as a regulatory factor rather than an essential component of its activity.