COST-ATP pretends to establish the role of intravesicular ATP in excitatory and inhibitory synapses of the central nervous system (CNS). Although, several laboratories have characterized the crucial interaction between ATP and catecholamines to permit its large accumulation in secretory vesicles of chromaffin cells, this crucial mechanism has not been studied in synaptic vesicles where high concentrations of neurotransmitters are needed for neuronal communication in the CNS. COST-ATP will combine the experience of the host laboratory in ATP as an accumulator of neurotransmitters in chromaffin cells and the TIRFM technology, and the ample experience of the researcher in cutting edge electrophysiological techniques in hippocampal neurons. The project will use electrophysiology, TIRFM, molecular biology and pharmacological tools to first discern the packagingbrole of vesicular ATP from its actions as neurotransmitter in central synapses using autaptic cultures of mouse hippocampal neurons. COST-ATP wants to explain why ATP is present inside of synaptic vesicles of almost all neurons. The main advantage of our approach is that we can modify the vesicular ATP by acting on the specific vesicular nucleotide carrier (VNUT) without affecting its cellular functions as the molecular energy. The consideration of the ATP, by its colligative properties, as a regulator of the neurotransmission, opens a new door in the neuronal communication. Given that its accumulation is mediated by VNUT a regulation of its activity could constitute a new pharmacological target for the treatment of neurological, psychiatric and cardiovascular diseases without involving membrane receptors.

In neurons, sites of Ca2+ influx and Ca2+ sensors are located within 20-50 nm, in subcellular “Ca2+ nanodomains”. Such tight coupling is crucial for the functional properties of synapses and neuronal excitability. Two key players act together in nanodomains, coupling Ca2+ signal to membrane potential: the voltage-dependent Ca2+ channels (VDCC) and the large conductance Ca2+ and voltage-gated K+ channels (BK). BK channels are characterized by synergistic activation by Ca2+ and membrane depolarization, but the complex molecular mechanism underlying channel function is not adequately understood. Information about the pore region, voltage sensing domain or isolated intracellular domains has been obtained separately using electrophysiology, biochemistry and crystallography. Nevertheless, the specialized behavior of this channel must be studied in the whole protein complex at the membrane in order to determine the complete range of structures and movements critical to its in vivo function. Using a combination of genetics, electrophysiology and spectroscopy, our group has measured for the first time the structural rearrangements accompanying whole BK channel activation at the membrane. From this unique position, our first goal is to fully determine the real time structural dynamics underlying the molecular coupling of Ca2+, voltage and activation of BK channels in the membrane environment, its regulation by accessory subunits and channel effectors. BK subcellular localization and role in Ca2+ nanodomains make these channels perfect candidates as reporters of local changes in [Ca2+] restricted to specific nanodomains close to the neuronal membrane. In our laboratory we have created fluorescent variants of the channel that report BK activity induced by Ca2+ binding, or Ca2+ binding and voltage. Our second aim in this proposal is to optimize and deploy this novel optoelectrical reporters to study physiologically relevant Ca2+-induced processes both in cellular and animal mode