31P-NMR Analysis of Synaptic Vesicles. Status of ATP and Internal pH

Abstract

The status of ATP in cholinergic synaptic vesicles from the electric organ of Torpedo marmoraia has been studied by 31P-NMR. In isolated vesicles, ATP was found to be the only major phosphate-containing constituent. The chemical shifts and the linewidths of the P, and Pp resonances are different from those of uncompartmented ATP added to the suspension. The spectrum of intact electric tissue contains two sets of ATP resonances; one of them belongs to vesicular ATP as identified by its linewidths and chemical shifts.Both spectral characteristics of vesicular ATP, linewidths and chemical shifts, could be explained by an exchange mechanism, which also occurs in model solutions containing acetylcholine, ATP and Mg2+ at a pH around 5.5.We therefore conclude that the cholinergic synaptic vesicles store ATP together with acetylcholine and Mg2 ' essentially in free solution at an acidic pH. Since the chemical shift of the P, resonance of ATP is largely determined by factors other than pH, the concept of its use as an indicator for the internal pH has to be modified in the case of these organelles.The application of NMR to secretory organelles has yielded a wealth of information regarding the internal organization of these structures [I -41. The secretory vesicles of nerve terminals, the synaptic vesicles, store and release transmitter and therefore play a central role in synaptic transmission. For an understanding of their function it is necessary to know the organization of the core and the physical status of the components. The only source of synaptic vesicles homogeneous with respect to transmitter content and available in sufficient quantity are the purely cholinergic electromotor nerve terminals innervating the electric organs of Torpedinidae. The chemical composition of highly purified vesicle fractions from this source has been determined and the membrane proteins have been analysed [S -71. The vesicles behave like highly hydrated structures in which up to 65% of the water is osmotically active and a further 7 % is bound to readily diffusible solutes [8]. The vesicular cores do not contain soluble proteins like catecholamine-storing chromaffine granules [5]. The main storage products are acetylcholine and ATP. They are present at concentrations which, if they behaved as ideal solutes would generate an osmotic pressure considerably in excess of that of the surrounding cytoplasm [7,9] ; therefore some immobilization or complex formation may be expected.Recently, we have shown by 'H-NMR spectroscopy that the transmitter in these vesicles, acetylcholine, is stored in a dissolved form. In addition, 'H-NMR is able to distinguish compartmented from uncompartmented acetylcholine and to follow its release from the compartmented state [lo].Here, we apply 31P-NMR to isolated vesicles and to intact electric tissue. From the chemical shifts and the linewidths of the P,, Po and P, resonances of intravesicular ATP, the physical state of the stored ATP and the intravesicular pH are deduced. The interpretation is based on the sim...

Characterization of a Mg2+-ATPase and a proton pump in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata

“…These observations are in agreement with the finding using a chemical method [6] or 31P-NMR [7] that the pH within vesicles is acidic relative to the neutral external medium and has a value around 5. 2-5.7.…”

“…These observations are in agreement with the finding using a chemical method [6] or 31P-NMR [7] that the pH within vesicles is acidic relative to the neutral external medium and has a value around 5.2-5.7. It was also shown [8, 9, 101 that treatment of isolated vesicles with ionophores causes release of acetylcholine providing additional evidence that an electrochemical gradient across the membrane is necessary for transmitter storage.…”

“…The released ATP in the presence of external Mg2+ then provides a continuous supply for ATP-driven ['4C]methylamine accumulation, so that no extra external ATP is needed. A comparison of the equilibration values in the presence and absence of ammonia suggests that intact vesicles have a higher initial ApH than ghosts, probably reflecting the capacity of the particle to retain ion gradients after isolation for a relatively long time [7]. If vesicle pellets after hypo-osmotic shock are resuspended in the iso-osmolar buffer used for vesicle isolation which is hyper-osmolar relative to the lysis medium, ATP-dependent methylamine uptake is negligible.…”

“…upon storage they lose these constitutents slowly (half-life approx. 36 h, [7]). The released ATP in the presence of external Mg2+ then provides a continuous supply for ATP-driven ['4C]methylamine accumulation, so that no extra external ATP is needed.…”

Specific sorting of the a1 isoform of the V-H+ATPase a subunit to nerve terminals where it associates with both synaptic vesicles and the presynaptic plasma membrane

“…The large electrochemical H + gradient generated by this enzyme [pH 5.2-5.5 inside the synaptic vesicles (Michaelson and Angel, 1980;Füldner and Stadler, 1982)] is used by specific vesicular transporters to accumulate the neurotransmitter. In addition to this well established role in neurotransmitter storage, V-ATPase could also participate in the constitution of a fusion pore involved in neurotransmitter release (Morel et al, 2001) and membrane fusion (Peters et al, 2001).…”